KR20140097143A - Compositions and methods for silencing aldehyde dehydrogenase - Google Patents

Abstract

The invention provides compositions comprising a therapeutic nucleic acid, such as an interfering RNA (e. G., A dsRNA, such as siRNA) targeting the expression of an aldehyde dehydrogenase (ALDH) gene, a therapeutic nucleic acid of one or more (e. G., Cocktail) (For example, to treat alcohol poisoning in humans), and a method of delivering and / or administering lipid particles.

Description

Technical Field [0001] The present invention relates to compositions and methods for silencing aldehyde dehydrogenases,

Cross-reference to related application

This application claims the benefit under 35 USC §119 (e) of U.S. Provisional Patent Application No. 61 / 599,238, filed February 15, 2012, and U.S. Provisional Patent Application 61 / 543,700, filed October 5, 2011, The contents of which are incorporated herein by reference in their entirety.

References to Sequence Listing

The sequence listing associated with this application is provided in text format instead of paper form, and is incorporated herein by reference. The name of the text file containing the sequence list is TEKM_074_02WO_ST25.txt. The text file is 12 KB, created on October 4, 2012, and electronically submitted via EFS-Web.

Alcoholism is an addiction or dependence on the consumption of excessive amounts of alcoholic beverages, such as beer, wine and distilled spirits. Alcoholism is sometimes referred to as alcohol abuse or alcohol dependence.

The biological mechanisms underlying alcoholism are unclear, but risk factors include stress, mental health problems and genetic predisposition. Long - term alcohol abuse produces physiological changes in the brain that cause alcohol withdrawal syndrome when alcohol consumption stops. Alcohol damages almost all body organs, and alcoholics are at risk of medical and psychological illnesses.

Treatment of alcoholism is problematic and usually involves alcohol detoxification to stop the alcoholic drinkers from stopping drinking. Neuroactive drugs such as benzodiazepines can be used to manage alcohol withdrawal symptoms. Post-medical care, such as psychotherapy, is generally required to maintain this week.

Disulfiram is a drug that causes acute susceptibility to ingested alcohol (ethanol) and is sometimes used in the treatment of chronic alcoholism. The alcohol is broken down in the liver by the enzyme alcohol dehydrogenase to acetaldehyde, which is then converted to acetic acid by the enzyme acetaldehyde dehydrogenase. Disulfiram blocks the enzyme acetaldehyde dehydrogenase. Thus, disulfiram is used in such a way that the concentration of acetaldehyde in the blood of humans consuming alcohol is substantially higher than that found in the blood of a person who consumes the same amount of alcohol in the absence of disulfiram (for example, 5 to 10 times higher). Acetaldehyde is one of the major causes of "hangover" symptoms, and therefore disulfiram consumption causes a serious negative reaction to alcohol. Symptoms include blushing of the skin, increased heart rate, shortness of breath, nausea, vomiting, throbbing headache, visual anomalies, delirium, somnolence, and circulatory insufficiency.

However, disulfiram has clinical limitations due to poor patient adherence and a wide range of side effects such as drowsiness, headache and less frequent neurotoxicity. Disulfiram is generally administered daily to be effective, and therefore, it is easy for the patient to stop taking the drug.

Accordingly, there is a continuing need for compositions and methods for inhibiting, reducing and / or eliminating the activity of acetaldehyde dehydrogenase associated with alcohol metabolism in mammals, especially humans. Such compositions and methods can be used, for example, in the treatment of alcoholism. In particular, the use of acetaldehyde dehydrogenase for a significantly longer period of time (e. G., Weeks or months) than the shelf life of disulfiram so as to make it difficult to stop alcohol abhorrence therapy after the human subject has consumed such relatively long-acting ALDH inhibitor There is a need for compositions and methods for inhibiting, reducing and / or eliminating the activity of a genase. There is also a particular need for compositions and methods that inhibit, reduce and / or eliminate the activity of acetaldehyde dehydrogenase, with fewer or lesser side effects than disulfiram.

A brief overview of the invention

As will be more fully described herein, acetaldehyde dehydrogenase is a member of the broader class of aldehyde dehydrogenase (ALDH) enzymes. A member of the ALDH family, which is believed primarily responsible for the conversion of acetaldehyde to acetic acid in human liver, is the aldehyde dehydrogenase 2 (ALDH2) enzyme, but other isoforms of ALDH, such as ALDH1, I am involved. It is an object of the present invention to provide compositions and methods for inhibiting the expression of one or more ALDH enzymes, particularly one or more genes encoding ALDH2 enzymes. Inhibition is mediated through RNA interference mechanisms. Thus, for example, the compositions and methods of the present invention inhibit or block the conversion of acetaldehyde to acetic acid, thereby increasing the amount of acetaldehyde in the body of a person who has consumed the alcohol, and consequently the presence of acetaldehyde in the body It is useful for treating alcoholism in humans by enhancing adverse effects (e. G., Headache and nausea).

Accordingly, the present invention provides compositions comprising therapeutic nucleic acids, such as interfering RNAs (e. G., DsRNA, such as siRNA) targeting the expression of an aldehyde dehydrogenase (ALDH) gene, a composition comprising one or more (e.g., cocktail ), A method of preparing lipid particles, and a method of delivering and / or administering lipid particles (e.g., to treat alcohol intoxication).

More particularly, the invention provides compositions comprising unmodified and chemically modified interfering RNA (e.g., siRNA) molecules that inhibit or silence ALDH gene expression. The present invention also provides serum-stable nucleic acid-lipid particles (e. G., Liposomes) comprising one or more (e.g., cocktails) of interfering RNA (e. G., SiRNA), cationic lipids and non- For example, SNALP) and its preparation, which may additionally comprise a conjugated lipid that inhibits aggregation of the particles. Examples of interfering RNA molecules include, but are not limited to, double stranded RNA (dsRNA) such as siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, pre-miRNA, and combinations thereof.

In one aspect, the invention provides an interfering RNA that targets ALDH gene expression wherein the interfering RNA comprises a sense strand and a complementary antisense strand, wherein the interfering RNA comprises a double stranded region of about 15 to about 60 nucleotides in length . In certain embodiments, the invention provides a combination of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more interfering RNA molecules targeting the same and / or different regions of the ALDH genome For example, a cocktail). The interfering RNA of the present invention can inhibit or silence ALDH gene expression in vitro and in vivo.

For example, a non-limiting example of an ALDH2 transcript sequence that can be used in the design of siRNA molecules that inhibit ALDH2 gene expression is provided in Genbank (www.ncbi.nlm.nih.gov/genbank) under accession number NM_000690 .3 (gene ID 217, isoform 1) and NM 001204889.1 (gene ID 217, isoform 2).

For example, a non-limiting example of an ALDHl transcript sequence that can be used in the design of siRNA molecules that inhibit ALDHl gene expression is disclosed in Genbank (www.ncbi.nlm.nih.gov/genbank) under accession number NM_000689.4 ALDH1A1, gene ID 216), and NM_000692.4 (ALDH1B1, gene ID 219).

In another aspect, the invention provides an interfering RNA targeting an aldehyde dehydrogenase (ALDH) gene expression, wherein the interfering RNA comprises a sense strand and a complementary antisense strand, wherein the interfering RNA is from about 15 to about 60 Double-stranded regions of single nucleotide length. In certain embodiments, the invention provides a combination of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more interfering RNA molecules targeting the same and / or different regions of the ALDH gene For example, a cocktail). The interfering RNA of the present invention can inhibit or completely silence ALDH gene expression in vitro and in vivo.

Each interfering RNA sequence present in the composition of the present invention may be independently located at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 More than two modified nucleotides, such as 2 ' Ome nucleotides. Preferably, the uridine and / or guanine nucleotides are modified into 2'OMe nucleotides. In certain embodiments, each interfering RNA sequence present in a composition of the invention comprises at least one 2'OMe-uridine nucleotide and at least one 2'OMe-guanosine nucleotide in the sense and / or antisense strand.

Examples of double-stranded siRNA molecules useful in the practice of the invention to inhibit ALDH2 gene expression include double-stranded siRNA molecules (1 to 6) having the chemically modified sense and antisense strand sequences set forth in Table 4 of this Example 4 Numbering). The double-stranded siRNA molecule described in Table A of Example 4 is chemically modified by the presence of a 2'-O-methyl moiety on the ribonucleotide unit identified by the letter "m ". The invention also includes double-stranded siRNA molecules having the sense and antisense sequences set forth in Table A of Example 4, wherein the sense and antisense strands are not chemically modified. The invention also includes isolated single-stranded nucleic acid molecules having any one sense strand sequence (chemically modified or chemically unmodified) as set forth in Table A of Example 4. The invention also encompasses isolated single-stranded nucleic acid molecules having any one of the antisense strand sequences (chemically modified or chemically unmodified) set forth in Table A of Example 4.

The present invention also provides a pharmaceutical composition comprising one or a cocktail of molecules of interfering RNA (e. G., SiRNA) targeting ALDH gene expression, and a pharmaceutically acceptable carrier.

In another aspect, the invention provides nucleic acid-lipid particles targeting ALDH gene expression. Nucleic acid-lipid particles typically include one or more unmodified and / or modified interfering RNA, cationic lipid, and non-cationic lipid that silence ALDH gene expression. In certain instances, the nucleic acid-lipid particle further comprises a conjugated lipid that inhibits aggregation of the particles. In a preferred embodiment, the nucleic acid-lipid particle comprises at least one unmodified and / or modified interfering RNA silencing ALDH gene expression, a cationic lipid, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of the particles do.

In another embodiment, the interfering RNA molecules of the invention are fully encapsulated within nucleic acid-lipid particles (e. G., SNALP). In preparations involving cocktails of interfering RNAs, different types of interfering RNA molecules may be co-encapsulated within the same nucleic acid-lipid particle, or each type of interfering RNA species present within the cocktail may be encapsulated within its own particle .

The present invention also provides pharmaceutical compositions comprising nucleic acid-lipid particles and a pharmaceutically acceptable carrier.

The nucleic acid-lipid particles of the present invention are useful for the prophylactic or therapeutic delivery of interfering RNA (e. G., DsRNA) molecules that silence the expression of one or more ALDH genes (e. G., The ALDH2 gene). In some embodiments, the one or more interfering RNA molecules described herein are formulated as nucleic acid-lipid particles, and the particles are administered to a mammal (e.g., a human) that requires treatment. In certain instances, a therapeutically effective amount of the nucleic acid-lipid particle may be administered to prevent or treat alcohol intoxication in a mammal, e. G., A human. The nucleic acid-lipid particles of the present invention are particularly useful for targeting liver cells that are the sites of most ALDH2 gene expression in humans. Administration of the nucleic acid-lipid particles may be by any route known in the art such as, for example, oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, . In certain embodiments, the nucleic acid-lipid particles are administered systemically, for example, via the intestinal or parenteral route of administration.

In some embodiments, down-regulation of ALDH gene expression is determined by detecting ALDH RNA or protein levels in the biological sample from the mammal after the nucleic acid-lipid particle administration. In another embodiment, down-regulation of ALDH gene expression is determined by detecting ALDH mRNA or protein levels in the biological sample from the mammal after the nucleic acid-lipid particle administration. In certain embodiments, downregulation of ALDH or ALDH gene expression is detected by monitoring symptoms associated with alcohol withdrawal in a mammal after particle administration.

In another embodiment, the invention provides a method of introducing into a cell an interfering RNA that silences ALDH gene expression, comprising contacting the cell with a nucleic acid-lipid particle of the invention.

In another embodiment, the present invention provides a method of treating an ALDH gene in a mammal (e. G., A human), comprising the step of administering to a mammal in need of silencing of ALDH gene expression a nucleic acid- Lt; RTI ID = 0.0 > expression. ≪ / RTI >

In another aspect, the invention provides a method of treating and / or ameliorating at least one symptom associated with alcoholism in a human, comprising administering to a human a therapeutically effective amount of the nucleic acid-lipid particle of the present invention.

In yet another aspect, the invention provides a method of treating a mammal in need of such an inhibition of ALDH expression, comprising administering to said mammal a therapeutically effective amount of a nucleic acid-lipid particle of the present invention, RTI ID = 0.0 > human). ≪ / RTI >

In a further aspect, the present invention provides a method of preventing and / or treating alcohol addiction in a human, comprising administering a therapeutically effective amount of a nucleic acid-lipid particle of the present invention to a human, respectively.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description and drawings.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The interfering RNA (e. G., SiRNA) drug therapy described herein advantageously provides important new compositions and methods for treating alcoholism in humans. An embodiment of the invention may be administered, for example, once a week, once a week, or once every few weeks (e.g., once every 2, 3, 4, 5 or 6 weeks). It may be more difficult for an alcoholic to abstain from alcohol abstinence after consuming the composition of the present invention effective for inhibiting ALDH for days or weeks.

Moreover, the lipid particles described herein (e. G., SNALP) enable effective delivery of nucleic acid drugs, e. G. Interfering RNA, into the target tissues and cells within the body. The presence of lipid particles provides protection from neclease breakdown in the blood stream, permits preferential accumulation within target tissues, and provides a means of drug delivery into the cellular cytoplasm where the siRNA can perform its intended function of RNA interference to provide.

II . Justice

As used herein, the following terms have the meanings specified for them unless otherwise specified.

The term "aldehyde dehydrogenase" (abbreviated as ALDH) refers to an enzyme that catalyzes the oxidation (dehydrogenation) of an aldehyde (e.g., acetaldehyde) to a carboxylic acid (e.g., acetic acid). Aldehyde dehydrogenase 2 (ALDH2), an enzyme located in the mitochondria, which is thought to be primarily responsible for the conversion of acetaldehyde to acetic acid in humans, is a family of structurally and functionally related aldehyde dehydrogenases present in mammals .

The term "interfering RNA" or "RNAi" or "interfering RNA sequence ", as used herein, refers to an RNA molecule that, when the interfering RNA is in the same cell as the target gene or sequence (e.g., mediates degradation of mRNA complementary to the interfering RNA sequence (E.g., mature miRNA, ssRNAi oligonucleotide, ssDNAi oligonucleotide) or double stranded RNA (i.e., duplexes) capable of reducing or suppressing the expression of a target gene or sequence RNA, such as siRNA, dicer-substrate dsRNA, shRNA, aiRNA, or pre-miRNA). Thus, an interfering RNA represents a single stranded RNA complementary to a double stranded RNA formed in a target mRNA sequence, or by two complementary strands or by a single self-complementary strand. The interfering RNA may have substantial or complete identity to the target gene or sequence, or it may comprise a region of mismatch (i.e., a mismatch motif). The sequence of the interfering RNA may correspond to a full-length target gene, or a subsequence thereof. Preferably, the interfering RNA molecules are chemically synthesized.

The interfering RNA can be a "small interfering RNA" or "siRNA", eg, about 15-60, 15-50, or 15-40 (duplex) nucleotide lengths, more typically about 15-30, Preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (for example, each of the double-stranded siRNAs) The complementarity sequence is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or 21-23 nucleotides And the double-stranded siRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferably about 18-22, 19-20, 21 base pairs long). siRNA duplexes can comprise from about 1 to about 4 nucleotides or from about 3 to about 3 nucleotides 3 'overhang, and 5' phosphate termini. Examples of siRNAs include, but are not limited to, double-stranded polynucleotide molecules assembled from two distinct strands of molecules (where one strand is a sense strand and the other is a complementary antisense strand); Double-stranded polynucleotide molecules assembled from a single strand of molecules, wherein the sense and antisense regions are joined by a nucleic acid-based or non-nucleic acid-based linker; Double-stranded polynucleotide molecules having a hairpin secondary structure with self-complementary sense and antisense regions; And a circular single-stranded polynucleotide molecule having two or more loop structures, wherein the circular polynucleotide is treated in vivo or in vitro to form an active double-stranded siRNA molecule And the like).

Preferably, the siRNA is chemically synthesized. siRNA also has a Can be generated by cleavage of longer dsRNAs (e. G., DsRNAs of about 25 nucleotide lengths longer) using E. coli RNase III or a dicer. These enzymes treat dsRNAs with biologically active siRNAs (see, for example, Yang et al., Proc. Natl. Acad. Sci. USA, 99: 9942-9947 (2002); Calegari et al., Proc (Kawasaki et al., Nucleic Acids Res., 10: 1): < RTI ID = 0.0 & (Robertson et al., J. Biol. Chem., 243: 82 (1968)), which is incorporated herein by reference in its entirety); [Knight et al., Science, 293: 2269-2271 ). Preferably, the dsRNA is at least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotides in length. The dsRNA can be as long as 1000, 1500, 2000, 5000 nucleotides in length or more. A dsRNA can encode a whole gene transcript or a partial gene transcript. In certain cases, the siRNA may be coded by the plasmid (e. G., Be transcribed as a sequence that automatically folds into a duplex with a hairpin loop).

As used herein, the term "mismatch motif or" mismatch region "refers to a portion of an interfering RNA (e.g., siRNA) sequence that is not 100% complementary to its target sequence. Mismatch regions may have 2, 3, 4, 5, 6, or more mismatch regions. The mismatch regions may be contiguous, or may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, The mismatch motif or region may comprise a single nucleotide or may comprise 2, 3, 4, 5 or more nucleotides.

The phrase "inhibiting the expression of a target gene" refers to the ability of the inventive interfering RNA (e.g., siRNA) to silence, reduce or inhibit the expression of a target gene (e.g., an ALDH gene). To examine the degree of gene silencing, a test sample (e.g., a biological sample from a subject organism expressing the target gene, or a sample of cells in a culture fluid expressing the target gene) is either silenced or reduced in expression of the target gene (E. G., SiRNA). The expression of a target gene in a test sample can be measured in a control sample not contacted with an interfering RNA (e. G., SiRNA) (e. G., A biological sample of the organism of interest expressing the target gene, Sample) with the expression of the target gene. A 100% value can be assigned to a control sample (e. G., A sample expressing the target gene). In certain embodiments, the silencing, inhibiting, or reducing expression of the target gene is less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60% , 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 10%, 5%, or 0%. Suitable assays include, but are not limited to, techniques well known to those skilled in the art such as, but not limited to, dot blotting, Northern blot, in situ hybridization, ELISA, immunoprecipitation, RTI ID = 0.0 > mRNA < / RTI >

An "effective amount" or "therapeutically effective amount" of a therapeutic nucleic acid, such as interfering RNA, is an amount sufficient to produce an inhibition of the expression of a target sequence relative to a desired effect, for example, a normal expression level detected in the absence of interfering RNA. In certain embodiments, the inhibition of the expression of the target gene or target sequence is inhibited by about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60% , 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0%. Suitable assays for measuring expression of a target gene or target sequence include, but are not limited to, techniques known to those skilled in the art such as, for example, dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, But are not limited to, testing of the level of protein or mRNA used.

Quot; reducing, "" reducing, " or" reducing, "an immune response by an interfering RNA means a detectable reduction in the immune response to a given interfering RNA (e. It is intended. The amount of reduction of the immune response by the modified interfering RNA can be determined relative to the level of the immune response in the presence of the unmodified interfering RNA. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or more than the detected immune response in the presence of unmodified interfering RNA. , 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or even lower. The reduction in the immune response to the interfering RNA is usually due to a decrease in cytokine production (e.g., IFNγ, IFNα, TNFα, IL-6, or IL-12) by responder cells in vitro after administration of the interfering RNA, Or by reduction of cytokine production in the serum of a mammalian subject.

As used herein, the term "responding cell" refers to a cell, preferably a mammalian cell, that produces a detectable immune response when contacted with immunostimulatory interfering RNA, such as unmodified siRNA. Exemplary responder cells include, for example, dendritic cells, macrophages, peripheral blood mononuclear cells (PBMCs), spleen cells, and the like. Detectable immune responses include, for example, cytokines or growth factors such as TNF- [alpha], IFN- [alpha], IFN- [beta], IFN-y, IL-1, IL-2, IL-3, IL- , IL-6, IL-10, IL-12, IL-13, TGF, and combinations thereof. Detectable immune responses also include induction of interferon-induced proteins using, for example, tetracycline peptide repeat 1 (IFITl) mRNA.

"Substantial identity" refers to a sequence that hybridizes to a reference sequence under stringent conditions, or a sequence having the percent identity specified over the specified region of the reference sequence.

The phrase "stringent hybridization conditions" refers to conditions under which the nucleic acid hybridizes to its target sequence, but not to other sequences, usually in a complex mixture of nucleic acids. Strict conditions are sequence-dependent and will be different in different environments. Longer sequences hybridize specifically at higher temperatures. An extensive introduction to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes (1993). In general, stringent conditions are selected to be about 5-10 ° C lower than the thermal melting point (T _m ) for a specific sequence at a defined ionic strength pH. T _m is the temperature at which 50% of the probes complementary to the target are hybridized to the target sequence at equilibrium (50% of the probe is occupied in equilibrium at T _m when the target sequence is present in excess) Strength, pH, and nucleic acid concentration). Strict conditions can also be achieved by addition of a destabilizing agent such as formamide. For selective or specific hybridization, the positive signal is at least 2 times background, preferably 10 times background hybridization.

Exemplary stringent hybridization conditions may be as follows: 50% formamide, 5x SSC, and 1% SDS, incubating at 42 ° C, or incubating at 5x SSC, 1% SDS, 65 ° C followed by 0.2x SSC, and 0.1% Gt; 65 C < / RTI > within SDS. For PCR, a temperature of about 36 ° C is typical for low stringency amplification, but the annealing temperature can vary from about 32 ° C to 48 ° C depending on the primer length. For high stringency PCR amplification, a temperature of about 62 ° C is typical, but high stringency annealing temperatures can range from about 50 ° C to about 65 ° C, depending on primer length and specificity. Typical cycling conditions for both high and low stringency amplification are denaturing periods of 90 ° C-95 ° C for 30 sec to 2 min, annealing times lasting 30 sec to 2 min, and extension times of 72 ° C for 1 to 2 min . Protocols and guidelines for low and high stringency amplification reactions are described, for example, in Innis et al., PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y. (1990).

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if their encoding polypeptides are substantially identical. This occurs, for example, when a copy of the nucleic acid is generated using the maximum codon degeneracy allowed by the genetic code. In such cases, the nucleic acids are usually hybridized under moderately stringent hybridization conditions. Exemplary "Moderately stringent hybridization conditions" include hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37 ° C, and washing in 1X SSC at 45 ° C. Positive hybridization is at least twice the background. One skilled in the art will readily recognize that separate hybridization and washing conditions can be used to provide conditions of similar stringency. Additional guidelines for determining the hybridization parameters are provided in numerous references, for example, Current Protocols in Molecular Biology, Ausubel et al., Eds.

The term "substantially the same" or "substantial identity" in the context of two or more nucleic acids refers to the maximum correspondence on a designated region, or comparison window, using one of the following sequence comparison algorithms or by manual alignment and visual inspection (I.e., at least about 60%, preferably at least about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over the specified region ) ≪ / RTI > nucleotides. The above definitions also refer to the complement of the sequence when the context indicates. Preferably, substantial identity is present over a region of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in length.

For sequence comparison, usually one sequence acts as a reference sequence and the test sequences are compared against it. When using a sequence comparison algorithm, enter test sequences and reference sequences into a computer, specify subsequence coordinates if necessary, and specify sequence algorithm program parameters. You can use the default program parameters, or you can specify separate parameters. The sequence comparison algorithm then calculates the% sequence identity for the test sequence relative to the reference sequence based on the program parameters.

As used herein, " comparison window " includes references to one of many contiguous positions selected from the group consisting of about 5 to about 60, usually about 10 to about 45, more usually about 15 to about 30, Wherein the sequence can be compared to a reference sequence of the same number of consecutive positions after the two sequences are optimally aligned. Sequence alignment methods for comparison are known in the art. Optimal alignment of sequences for comparison is described, for example, in Smith and Waterman, Adv. Appl. Math., 2: 482 (1981)] by the local homology algorithm of Needleman and Wunsch, J. Mol. Biol., 48: 443 (1970), by Pearson and Lipman, Proc. Natl. Acad. Sci. (Wisconsin Genetics Software Package; Genetics Computer Group, Madison Scientific Drive 575, Wisconsin, USA), by a search method for similarity of these algorithms, (See, for example, GAP, BESALDHIT, FASTA, and ALDHASTA in the US), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology, Ausubel et al. .

Non-limiting examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res., 25: 3389-3402 (1977) and Altschul et al., J. Mol. Biol, 215: 403-410 (1990). BLAST and BLAST 2.0 are used with the parameters described herein to determine the% sequence identity for the nucleic acids of the present invention. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Another example is the Needleman-Wunsch algorithm for sorting an overall alignment algorithm, e. G., Protein or nucleotide (e. G., RNA) sequences, to determine percent sequence identity.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90: 5873-5787 (1993)). One measure of the similarity provided by the BLAST algorithm is the minimum sum probability (P (N)), which provides an indication of the probability that a match between two nucleotide sequences will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence when the minimum sum probability in the comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

The term "nucleic acid" when used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in the form of single-stranded or double-stranded forms, including DNA and RNA. The DNA may be, for example, an antisense molecule, a plasmid DNA, a pre-condensed DNA, a PCR product, a vector (P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, a chromosomal DNA, Derivatives and combinations thereof. The RNA may be a small interfering RNA (siRNA), dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA May be present in the form of a combination thereof. Nucleic acids include nucleic acid containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring and non-naturally occurring and have binding characteristics similar to reference nucleic acids. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methyl ribonucleotides, and peptide- . Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides having binding characteristics similar to reference nucleic acids. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes a conservative modified variant (e. G., Degenerate codon substitution), an allelic genome, an ortholog, a SNP, and a sequence explicitly indicated with a complementary sequence do. Specifically, degenerative codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with a mixed-base and / or deoxyinosine residue (Batzer et al. (Rossolini et al., Mol. Cell. Probes, 8: 309-5081 (1991)]; [Ohtsuka et al., J. Biol. Chem., 260: 2605-2608 91-98 (1994)). "Nucleotides" contain sugar deoxyribose (DNA) or ribose (RNA), base, and phosphate groups. The nucleotides are linked together through a phosphate group. The term "base" refers to purines and pyrimidines that additionally include the natural compound adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and the new reactivity, such as, but not limited to, amine, alcohol, thiol, carboxylate and alkyl halide And synthetic derivatives of purine and pyrimidine, including, but not limited to, those that place the group.

The term "gene" refers to a nucleic acid (e. G., DNA or RNA) sequence comprising a partial length or full length coding sequence necessary for the production of a polypeptide or a precursor polypeptide.

"Gene product" when used herein refers to a product of a gene, such as an RNA transcript or polypeptide.

The term "lipid" refers to a group of organic compounds which are not limited to, but are not limited to, esters of fatty acids and which are insoluble in water but soluble in many organic solvents. They are broadly divided into at least three classes: (1) "simple lipids" including fats and oils and also wax; (2) "complex lipids" including phospholipids and glycolipids; And (3) "derived lipids" such as steroids.

The term "lipid particles" includes lipid formulations that can be used to deliver therapeutic nucleic acids (e.g., interfering RNA) to a target site of interest (e.g., cells, tissues, organs, etc.). In a preferred embodiment, the lipid particle of the present invention is a nucleic acid-lipid particle, which is usually formed from a cationic lipid, a non-cationic lipid, and optionally a lipid that interferes with the aggregation of the particles. In another preferred embodiment, the therapeutic nucleic acid (e. G., Interfering RNA) can be encapsulated within the lipid portion of the particle, thus being protected from enzymatic degradation.

As used herein, the term "SNALP" refers to stable nucleic acid-lipid particles. SNALP refers to particles made from lipids (e. G., Cationic lipids, non-cationic lipids, and optionally lipids that interfere with the aggregation of particles), wherein the nucleic acid Encapsulated. In certain cases, SNALP may show extended blood flow life after intravenous (iv) injection and may accumulate at a distal site (e.g., a site physically separated from the site of administration), and gene expression at these distal sites It is very useful for whole body use because it can mediate the silence of. Nucleic acid can be complexed with the cofactor and encapsulated within the SNALP, as described in PCT Publication No. WO 00/03683, the disclosure of which is incorporated herein by reference in its entirety.

The lipid particles (e.g., SNALP) of the present invention typically have an average diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, From about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 nm to about 90 nm, from about 80 nm to about 90 nm, 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm , 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm and is substantially non-toxic. In addition, when the nucleic acid is present in the lipid particles of the present invention, it is resistant to nuclease degradation in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Patent Nos. 20040142025 and 20070042031, the entire contents of which are incorporated herein by reference in their entirety.

As used herein, "lipid encapsulated" may refer to a lipid particle that provides a therapeutic nucleic acid, e.g., an interfering RNA (e.g., siRNA), in a complete encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid (e. G., Interfering RNA) is fully encapsulated within the lipid particle (e. G., To form SNALP or other nucleic acid-lipid particles).

The term "lipid conjugate" refers to a conjugated lipid that inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to, PEG-lipid conjugates such as PEG (e.g., PEG-DAA conjugate) conjugated to a dialkyloxypropyl, PEG (e.g., PEG-DAG conjugate) conjugated to diacylglycerol, (E. G., POZ-lipid conjugates (e. G., ≪ RTI ID = 0.0 > POZ- (E.g., U.S. Provisional Patent Application Serial No. 61 / 294,828 filed January 13, 2010 and U.S. Provisional Patent Application Serial No. 61 / 295,140 filed January 14, 2010), polyamide oligomers (e.g., ATTA- Lipid conjugates), and mixtures thereof. Additional examples of POZ-lipid conjugates are described in PCT Publication WO 2010/006282. PEG or POZ can be directly conjugated to the lipid or can be linked to the lipid via the linker moiety. For example, any suitable linker moiety can be used to link PEG or POZ to lipids, including ester-free linker moieties and ester-containing linker moieties. In certain preferred embodiments, ester-free linker moieties such as amides or carbamates are used. The disclosures of each of these patents are incorporated herein by reference in their entirety for all purposes.

The term "amphiphilic lipid" refers, in part, to any suitable material for which the hydrophobic portion of the lipid material is oriented towards the hydrophobic phase while the hydrophilic portion is for culturing towards the aqueous phase. Hydrophilic characteristics are derived from the presence of polar or charged groups such as carbohydrates, phosphates, carboxyls, sulfato, amino, sulfhydryls, nitro, hydroxyls, and other like groups. Hydrophobicity can be imparted by the inclusion of long-chain saturated and unsaturated aliphatic hydrocarbon groups and non-polar groups including, but not limited to, those groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group (s). Examples of amphiphilic compounds include, but are not limited to, phospholipids, amino lipids, and sphingolipids.

Representative examples of phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioloylphosphatidylcholine, ≪ / RTI > phosphatidylcholine, diphosphatidylcholine, and dirinoleoylphosphatidylcholine. Other compounds lacking phosphorus, such as sphingolipids, glycosylation families, diacylglycerols, and? -Acyloxysaccharides, are also within the group designated as amphipathic lipids. In addition, the amphiphilic lipids described above may be mixed with other lipids, including triglycerides and sterols.

The term "neutral lipid" refers to any number of lipid species present in uncharged or neutral zwitterion form at the selected pH. At physiological pH, such lipids include, for example, diacyl phosphatidylcholine, diacyl phosphatidyl ethanolamine, ceramides, sphingomyelin, cephalin, cholesterol, cervuloside, and diacylglycerol.

The term "non-cationic lipid" refers to any other neutral lipid or anionic lipid as well as any amphipathic lipid.

The term "anionic lipid" refers to any lipid negatively charged at physiological pH. These lipids include phospholipids such as phosphatidyl glycerol, cardiolipin, diacyl phosphatidylserine, diacyl phosphatidic acid, N-dodecanoyl phosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, , Palmitoylaylyl phosphatidylglycerol (POPG), and other anionic modifiers linked to neutral lipids.

The term "hydrophobic lipid" refers to a compound having a non-polar group, including, but not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and those groups optionally substituted with one or more aromatic, cycloaliphatic, or heterocyclic group (s). Suitable examples include, but are not limited to, diacyl glycerol, dialkyl glycerol, N, N-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2- Do not.

The terms "cationic lipid" and "amino lipid" are intended to encompass a mixture of one, two, three or more fatty acid or fatty alkyl chains and a pH- titratable amino head group (such as an alkylamino or dialkylamino head group) ≪ / RTI > and salts thereof. Cationic lipids are usually protonated (i.e., positively charged) at a pH below the pKa of the cationic lipids and are substantially neutral at pH above pKa. The cationic lipids of the present invention may also be referred to as titratable cationic lipids. In some embodiments, the cationic lipid comprises a protonatable tertiary amine amine (e. G., PH-titratable) head group; _C18 alkyl chains wherein each alkyl chain independently has from 0 to 3 (e.g., 0, 1, 2, or 3) double bonds; And an ether, ester, or ketal linkage between the head group and the alkyl chain. Such cationic lipids include, but are not limited to, DSDMA, DODMA, DLinDMA, DLenDMA, γ-DLenDMA, DLin-K-DMA, DLin-K- (Also known as MC2), and DLin-M-C3-DMA (also known as MC2), DL-M-C3-DMA, DLin-K-C4-DMA, DLen-C2K-DMA, (Also known as < RTI ID = 0.0 > MC3). ≪ / RTI >

The term "salt" includes any anionic and cationic complex, such as a complex formed between a cationic lipid and one or more anions. Non-limiting examples of anions include inorganic and organic anions such as hydride, fluoride, chloride, bromide, iodide, oxalate (e.g., hemioxalate), phosphate, The present invention relates to a process for the preparation of a compound of formula (I), wherein the compound is selected from the group consisting of a phosphate, a dihydrogen phosphate, an oxide, a carbonate, a bicarbonate, a nitrate, a nitrite, a nitrite, a bisulfite, a sulfide, But are not limited to, but are not limited to, talc, borate, formate, acetate, benzoate, citrate, tartrate, lactate, acrylate, polyacrylate, fumarate, maleate, itaconate, glycolate, gluconate, , Ascorbate, salicylate, polymethacrylate, perchlorate, chlorate, chlorite, hypochlorite, bromine But are not limited to, peroxides, peroxides, peroxides, peroxides, peroxides, peroxides, peroxides, peroxides, peroxides, hypobromites, iodates, alkylsulfonates, arylsulfonates, arsenates, arsenates, chromates, dichromates, cyanides, cyanates, thiocyanates, , And mixtures thereof. In certain embodiments, the salt of the cationic lipids disclosed herein is a crystalline salt.

The term "alkyl" includes straight or branched, non-cyclic or cyclic, saturated aliphatic hydrocarbons containing 1 to 24 carbon atoms. Representative saturated straight chain alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl and the like, while saturated branched alkyls include but are not limited to isopropyl, sec- Butyl, tert-butyl, isopentyl and the like. Representative saturated cyclic alkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, while unsaturated cyclic alkyls include, but are not limited to, cyclopentenyl, cyclohexenyl, and the like.

The term "alkenyl" includes alkyl as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyl includes both cis and trans isomers. Representative straight chain and branched alkenyl groups include but are not limited to ethylenyl, propenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, Butenyl, 2,3-dimethyl-2-butenyl, and the like.

The term "alkynyl" includes any alkyl or alkenyl as defined above which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl and the like.

The term "acyl" includes any alkyl, alkenyl, or alkynyl in which carbon at the attachment site is substituted with an oxo group as defined below. The following are non-limiting examples of acyl groups: -C (= O) alkyl, -C (= O) alkenyl, and -C (= O) alkynyl.

The term "heterocycle" means a 5- to 7-membered ring containing 1 to 2 heteroatoms independently selected from saturated, unsaturated, or aromatic and being selected from nitrogen, , Wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, the nitrogen heteroatom may optionally be quatemized, and any of said heterocycles includes a bicyclic ring fused to the benzene ring. The heterocycle may be attached via any heteroatom or carbon atom. The heterocycle may be heteroaryl as defined below and heteroaryl as defined below such as morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl , Tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, etc. But are not limited to these.

The term "optionally substituted alkyl", "optionally substituted alkenyl", "optionally substituted alkynyl", "optionally substituted acyl", and "optionally substituted heterocycle" . ≪ / RTI > In the case of an oxo substituent (= O), two hydrogen atoms are replaced. In this regard, the substituent is selected from the group consisting of oxo, halogen, heterocycle, -CN, -OR ^x , -NR ^x R ^y , -NR ^x C (= O) R ^y , -NR ^x SO ₂ R ^y , ) R ^x , -C (= O) OR ^x , -C (═O) NR ^x R ^y , -SO _n R ^x , and -SO _n NR ^x R ^y where n is 0, 1, or 2 , R ^x and R ^y are the same or different and independently hydrogen, alkyl, or heterocyclyl, each alkyl and heterocyclyl substituent is one or more oxo, halogen, -OH, -CN, alkyl, -OR ^x, heteroaryl ^{cycle, -NR x R y, -NR x} C (= O) R y, -NR x SO 2 R y, -C (= O) R x, -C (= O) OR x, -C (= O ) NR ^x R ^y , -SO _n R ^x , and -SO _n NR ^x R ^y . The term "optionally substituted" when used before listing of substituents means that each substituent in the list may be optionally substituted as described herein.

The term "halogen" includes fluoro, chloro, bromo, and iodo.

The term " fusogenic "refers to the ability of lipid particles, such as SNALP, to fuse with membranes of cells. The membrane may be a plasma membrane, or a membrane surrounding an organelle, such as endosomes, nuclei, and the like.

As used herein, the term "aqueous solution" refers to a composition that includes wholly or partially water.

As used herein, the term "organic lipid solution" refers to a composition comprising an organic solvent wholly or partially having a lipid.

"Distal site" when used herein refers to physically discrete sites, which are not limited to adjacent capillary blood vessels, but instead include sites that are widely distributed throughout the organism.

Nucleic acid-lipid particles, such as those that are "stable in serum" with respect to SNALP, mean that the particles are not significantly degraded after exposure to serum or nuclease assays that will significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNAse assays, or RNAse assays.

"Systemic delivery" when used herein, refers to the delivery of lipid particles that cause broad biological distribution of an active agent, such as interfering RNA (e.g., siRNA), in an organism. Some administration techniques can cause systemic delivery of certain drugs, while others do not. Systemic delivery means that an effective amount, preferably a therapeutically effective amount of the agent, is exposed to most parts of the body. In order to obtain a broad biological distribution, it is generally necessary to administer the blood (for example, by first passage organ (liver, lung, etc.) or by rapid nonspecific cell binding) Life is required. Systemic delivery of lipid particles can be accomplished by any means known in the art including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, systemic delivery of lipid particles is accomplished by intravenous delivery.

"Local delivery", as used herein, refers to the delivery of an active agent, such as an interfering RNA (eg, siRNA), directly to a target site in an organism. For example, the medicament can be delivered locally by injection by direct injection into a diseased site, another target site, or a target organ such as liver, heart, pancreas, kidney, and the like.

The term "mammal" refers to any mammalian species such as a human, mouse, rat, dog, cat, hamster, guinea pig, rabbit,

The term " reticuloendothelial system "or" RES " refers to a portion of the immune system, including reticuloendothelial cells, including proliferating cells located within reticular connective tissue such as mononuclear cells and macrophages. These cells usually accumulate in the lymph nodes and spleen. The interstitial tissue of Kupffer cells and tissues is also part of RES. RES is divided into primary and secondary lymphoid organs. The primary ("central") lymphoid organs are the sites where RES cells are produced. RES cells are produced in bone marrow. The thymus is also included because it is the site required for T cell maturation. The secondary ("peripheral") lymphoid organs are the sites where the cells of the RES function. This includes lymph nodes, tonsils, spleen, and "MALT" (mucosa-associated lymphoid tissue). MALTs are further divided into "GALT" (bowel-associated lymphoid tissue) and "BALT" (bronchus-associated lymphoid tissue). Intercuspal cells act as part of the system but are not organized into tissues; Instead, the liver is dispersed through the oval shaped blood vessels. Small cells of the central nervous system (CNS) can be regarded as part of the RES. These are scavenger cells that proliferate in response to CNS damage.

III . Description of Embodiments

The present invention relates to therapeutic nucleic acids, such as interfering RNAs (e. G., DsRNA, such as siRNA) targeting the expression of an ALDH gene, in particular an ALDH2 gene, lipid particles comprising one or more therapeutic (e.g. cocktail) Methods of making lipid particles, and methods of delivering and / or administering lipid particles (e.g., for the treatment of alcohol poisoning in humans).

In one aspect, the invention provides an interfering RNA molecule targeting ALDH gene expression. Non-limiting examples of interfering RNA molecules include double stranded RNA that can mediate RNAi, such as siRNA, dicer-substrate dsRNA, shRNA, aiRNA, pre-miRNA, and mixtures thereof. In certain instances, the invention provides a composition comprising a combination (e.g., cocktail, pool, or mixture) of interfering RNAs targeting different regions of the ALDH gene. In certain instances, the interfering RNA (e. G., SiRNA) molecules of the invention can silence ALDH gene expression and / or inactivate ALDH or inhibit replication of ALDH in vitro or in vivo.

In certain embodiments, the invention provides an interfering RNA (e.g., siRNA) that silences ALDH gene expression wherein the interfering RNA comprises a sense strand and a complementary antisense strand, wherein the interfering RNA comprises about 15 to about 60 Nucleotide lengths (e.g., about 15-60, 15-30, 15-25, 19-30, 19-25, 20-60, 20-55, 20-50, 20-45, 20-40, 35, 20-30, 20-25, 21-30, 21-29, 22-30, 22-29, 22-28, 23-30, 23-28, 24-30, 24-28, 25-60, 25-55, 25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides in length, or about 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length).

In certain embodiments, the interfering RNA (e. G., SiRNA) of the present invention can comprise at least one, two, three, four, five, six, 7, 8, 9, or 10 or more modified nucleotides, such as 2'OMe nucleotides. Preferably, uridine and / or guanine nucleotides in the interfering RNA are modified to 2 ' Ome nucleotides. In certain cases, the interfering RNA contains 2'OMe nucleotides in both sense and antisense strands, and includes at least one 2'OMe-uridine nucleotide and at least one 2'OMe-guanosine nucleotide in the double-stranded region. In some embodiments, the sense and / or antisense strand of the interfering RNA can be, for example, modified (e. G., 2'OMe-modified) adenosine and / or modified , 2'OMe-modified) cytosine nucleotides.

In certain embodiments, an interfering RNA (e.g., siRNA) molecule of the invention comprises 3 ' overhangs of 1, 2, 3, or 4 nucleotides in either or both strands. In certain cases, the interfering RNA may contain at least one blunt end. In certain embodiments, the 3 'overhangs in one or both strands of the interfering RNA are each independently 1, 2, 3, or 4 modified and / or unmodified deoxythymidine ("t" ) Nucleotides, 1, 2, 3, or 4 modified (e.g., 2'OMe) and / or unmodified uridine ("U") ribonucleotides, or complementarity to a target sequence or a complementary strand thereof 1, 2, 3, or 4 modified (e. G., 2'OMe) and / or unmodified ribonucleotides or deoxyribonucleotides.

The invention also provides a pharmaceutical composition comprising one or more (e.g., cocktails) of the interfering RNA described herein and a pharmaceutically acceptable carrier.

In another aspect, the invention provides nucleic acid-lipid particles (e. G., SNALP) targeting ALDH gene expression. Nucleic acid-lipid particles (e. G., SNALP) typically include one or more (e. G., Cocktail) interfering RNAs, cationic lipids, and non-cationic lipids described herein. In certain cases, the nucleic acid-lipid particles (e. G., SNALP) further comprise a conjugated lipid that inhibits aggregation of the particles. Preferably, the nucleic acid-lipid particles (e. G., SNALP) comprise at least one (e. G., Cocktail) of the interfering RNA, cationic lipid, non-cationic lipid, It contains lipids.

In some embodiments, the interfering RNA (e. G., SiRNA) of the invention is fully encapsulated within nucleic acid-lipid particles (e. With respect to agents comprising interfering RNA cocktails, different types of interfering RNA species (e.g., interfering RNA compounds with different sequences) present within the cocktail can be co-encapsulated within the same particle, The types of interfering RNA species can be encapsulated within discrete particles. Interfering RNA Cocktails can be formulated within the particles described herein using a mixture of two or more individual interfering RNAs (each with a unique sequence) at the same, similar, or different concentrations or molar ratios. In one embodiment, the cocktail of the interfering RNA (corresponding to a plurality of interfering RNAs having different sequences) is formulated using the same, similar, or different concentration or molar ratio of each interfering RNA species, and different types of interfering RNA Co-encapsulated within the same particle. In another embodiment, each type of interfering RNA species present in the cocktail is encapsulated in different particles at the same, similar, or different interfering RNA concentration or molar ratio, and the particles thus formed (each containing a different interfering RNA payload ) May be administered separately (e.g., at different times, depending on the therapeutic formula), combined and administered together as a single unit dose (e. G., With a pharmaceutically acceptable carrier). The particles described herein are stable in serum, resistant to nuclease degradation, and substantially non-toxic to mammals, such as humans.

The cationic lipids in the nucleic acid-lipid particles (e.g., SNALP) of the present invention may include, for example, one or more cationic lipids of formula I-III as described herein or any other cationic lipid species. In one particular embodiment, the cationic lipid is 1,2-dirinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy- Dienol-4- (2-dimethylaminoethyl) - [1,3] - dihydroxy-N, N-dimethylaminopropane (DLin-K-C2-DMA), 2,2-dirinoleyl-4-dimethylaminomethyl- [1,3] -dioxolane Aminopropionate (DLin-M-C2-DMA), (6Z, 9Z, 28Z, 31Z) -heptatriaconta-6,9,28,31-tetraen- (DLin-M-C3-DMA), salts thereof, and mixtures thereof.

Non-cationic lipids within the nucleic acid-lipid particles (e.g., SNALP) of the present invention may include, for example, one or more anionic lipids and / or neutral lipids. In some embodiments, the non-cationic lipid comprises one of the following neutral lipid components: (1) a mixture of phospholipids and cholesterol or derivatives thereof; (2) cholesterol or a derivative thereof; Or (3) phospholipids. In certain preferred embodiments, the phospholipid comprises dipalmitoyl phosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), or a mixture thereof. In a particularly preferred embodiment, the non-cationic lipid is a mixture of DPPC and cholesterol.

A lipid conjugate in the nucleic acid-lipid particles (e.g., SNALP) of the present invention may inhibit aggregation of the particles and may include, for example, one or more lipid conjugates described herein. In one particular embodiment, the lipid conjugate comprises a PEG-lipid conjugate. Examples of PEG-lipid conjugates include, but are not limited to, PEG-DAG conjugates, PEG-DAA conjugates, and mixtures thereof. In certain embodiments, the PEG-DAA conjugate in the lipid particle is selected from the group consisting of a PEG-dimethyoxypropyl (C ₁₀ ) conjugate, a PEG-dilauryloxypropyl (C ₁₂ ) conjugate, a PEG-dimyristyloxypropyl (C ₁₄ ) A PEG-dipalmityloxypropyl (C ₁₆ ) conjugate, a PEG-distearyloxypropyl (C ₁₈ ) conjugate, or a mixture thereof. In another embodiment, the lipid conjugate comprises a POZ-lipid conjugate, such as a POZ-DAA conjugate.

In some embodiments, the present invention provides a method of treating an ALDH gene comprising: (a) contacting one or more (e.g., cocktail) interfering RNA molecules targeting ALDH gene expression; (b) at least one cationic lipid or salt thereof, accounting for from about 50 mole% to about 85 mole% of total lipid present in the particles; (c) at least one non-cationic lipid comprising from about 13 mol% to about 49.5 mol% of the total lipid present in the particles; And (d) at least one conjugated lipid that inhibits aggregation of the particles, accounting for from about 0.5 mol% to about 2 mol% of the total lipid present in the particles (for example, SNALP) .

In one aspect of this embodiment, the nucleic acid-lipid particle comprises (a) one or more (e. G., Cocktail) interfering RNA molecules targeting ALDH gene expression; (b) a cationic lipid or salt thereof, accounting for from about 52 mol% to about 62 mol% of the total lipid present in the particles; (c) a mixture of phospholipids and cholesterol or derivatives thereof, which comprises from about 36 mol% to about 47 mol% of the total lipid present in the particles; And (d) from about 1 mole percent to about 2 mole percent of the total lipid present in the particles. This embodiment of nucleic acid-lipid particles is generally referred to herein as a "1:57" formulation. In one particular embodiment, the 1:57 formulation comprises about 1.4 mol% PEG-lipid conjugate (e.g., PEG 2000-C-DMA), about 57.1 mol% cationic lipid (e.g., DLin- DMA) or a salt thereof, about 7.1 mole% DPPC (or DSPC), and about 34.3 mole% cholesterol (or derivatives thereof).

In another aspect of this embodiment, the nucleic acid-lipid particle comprises (a) one or more (e. G., Cocktail) interfering RNA molecules targeting ALDH gene expression; (b) a cationic lipid or salt thereof, accounting for from about 56.5 mol% to about 66.5 mol% of the total lipid present in the particles; (c) a cholesterol or derivative thereof accounting for from about 31.5 mol% to about 42.5 mol% of the total lipid present in the particles; And (d) from about 1 mole percent to about 2 mole percent of the total lipid present in the particles. This embodiment of nucleic acid-lipid particles is generally referred to herein as a "1:62" formulation. In one particular embodiment, the 1:62 formulation is free of phospholipids and comprises about 1.5 mole% PEG-lipid conjugate (e.g., PEG2000-C-DMA), about 61.5 mole% cationic lipid (e.g., DLin- K-C2-DMA) or a salt thereof, and about 36.9 mol% cholesterol (or a derivative thereof).

Additional embodiments involving 1:57 and 1:62 formulations are described in PCT Publication No. WO 09/127060 and United States Patent Application Publication No. US 2011/0071208 A1, the full text of which is incorporated herein by reference in its entirety.

In another embodiment, the present invention provides a method of treating an ALDH gene comprising: (a) contacting one or more (e. G., Cocktail) interfering RNA molecules targeting ALDH gene expression; (b) at least one cationic lipid or salt thereof accounting for from about 2 mol% to about 50 mol% of the total lipid present in the particles; (c) at least one non-cationic lipid comprising from about 5 mol% to about 90 mol% of the total lipid present in the particles; And (d) at least one conjugated lipid that inhibits aggregation of the particles, accounting for from about 0.5 mol% to about 20 mol% of the total lipid present in the particles (for example, SNALP) .

In one aspect of this embodiment, the nucleic acid-lipid particle comprises (a) one or more (e. G., Cocktail) interfering RNA molecules targeting ALDH gene expression; (b) a cationic lipid or salt thereof, accounting for from about 30 mole percent to about 50 mole percent of the total lipid present in the particles; (c) a mixture of phospholipids and cholesterol or derivatives thereof, accounting for from about 47 mol% to about 69 mol% of total lipid present in the particles; And (d) from about 1 mole percent to about 3 mole percent of the total lipid present in the particles. This embodiment of nucleic acid-lipid particles is generally referred to herein as a "2:40" formulation. In one particular embodiment, the 2:40 formulation comprises about 2 mole% PEG-lipid conjugate (e.g., PEG2000-C-DMA), about 40 mole% cationic lipid (e.g., DLin- DMA) or a salt thereof, about 10 mole% DPPC (or DSPC), and about 48 mole% cholesterol (or derivatives thereof).

In a further embodiment, the invention provides a kit comprising: (a) one or more (e. G., Cocktail) interfering RNA molecules targeting ALDH gene expression; (b) at least one cationic lipid or salt thereof, accounting for from about 50 mol% to about 65 mol% of the total lipid present in the particles; (c) at least one non-cationic lipid comprising from about 25 mol% to about 45 mol% of the total lipid present in the particles; And (d) at least one conjugated lipid that inhibits aggregation of the particles, accounting for from about 5 mol% to about 10 mol% of the total lipid present in the particles (for example, SNALP) .

In one aspect of this embodiment, the nucleic acid-lipid particle comprises (a) one or more (e. G., Cocktail) interfering RNA molecules targeting ALDH gene expression; (b) a cationic lipid or salt thereof, accounting for from about 50 mol% to about 60 mol% of the total lipid present in the particles; (c) a mixture of a phospholipid and a cholesterol or derivative thereof, wherein the phospholipid comprises from about 35 mol% to about 45 mol% of the total lipid present in the particle; And (d) from about 5 mole% to about 10 mole% of the total lipid present in the particles. This embodiment of nucleic acid-lipid particles is generally referred to herein as a "7:54" formulation. In certain cases, the non-cationic lipid mixture in the 7:54 formulation comprises (i) from about 5 mole percent to about 10 mole percent of the total lipid present in the particles; And (ii) from about 25 mol% to about 35 mol% of the total lipid present in the particles. In one particular embodiment, the 7:54 formulation comprises about 7 mole% PEG-lipid conjugate (e.g., PEG750-C-DMA), about 54 mole% cationic lipid (e.g., DLin- DMA) or a salt thereof, about 7 mole% DPPC (or DSPC), and about 32 mole% cholesterol (or derivatives thereof).

In another aspect of this embodiment, the nucleic acid-lipid particle comprises (a) one or more (e. G., Cocktail) interfering RNA molecules targeting ALDH gene expression; (b) a cationic lipid or salt thereof which comprises from about 55 mol% to about 65 mol% of the total lipid present in the particles; (c) a cholesterol or derivative thereof that comprises from about 30 mole% to about 40 mole% of the total lipid present in the particle; And (d) from about 5 mole% to about 10 mole% of the total lipid present in the particles. This embodiment of nucleic acid-lipid particles is generally referred to herein as a "7:58" formulation. In one particular embodiment, the 7:58 formulation is free of phospholipids and comprises about 7 mole% PEG-lipid conjugate (e.g., PEG750-C-DMA), about 58 mole% cationic lipid (e.g., DLin- K-C2-DMA) or a salt thereof, and about 35 mole% cholesterol (or a derivative thereof).

Additional embodiments involving 7:54 and 7:58 formulations are described in U.S. Patent Application Publication No. US 2011/0076335 A1, the full text of which is incorporated herein by reference in its entirety.

The present invention also provides pharmaceutical compositions comprising nucleic acid-lipid particles, such as SNALP, and a pharmaceutically acceptable carrier.

The nucleic acid-lipid particles (e. G., SNALP) of the present invention are useful for the therapeutic delivery of interfering RNAs (e. G. SiRNAs) that silence the expression of one or more ALDH genes. In some embodiments, the cocktail of the interfering RNA targeting different regions of the ALDH gene (e.g., overlapping and / or non-overlapping sequences) is formulated with the same or different nucleic acid-lipid particles, A treatment is administered to a mammal (e. In certain instances, a therapeutically effective amount of the nucleic acid-lipid particle can be administered to a mammal, for example, to treat alcohol poisoning in humans.

In certain embodiments, the invention provides methods for introducing one or more interfering RNA (e. G., SiRNA) molecules described herein into a cell by contacting the cell with a nucleic acid-lipid particle (e. G., A SNALP preparation) ≪ / RTI > In one particular embodiment, the cell is a reticuloendothelial cell (e. G., A mononuclear cell or a macrophage), a fibroblast, an endothelial cell, or a platelet cell.

In some embodiments, the nucleic acid-lipid particles (e. G., SNALP) described herein are administered by one of the following routes of administration: oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, , Subcutaneous, and intradermal. In certain embodiments, the nucleic acid-lipid particles are administered systemically, for example, via the intestinal or parenteral route of administration.

In certain embodiments, the nucleic acid-lipid particles (e. G., SNALPs) of the present invention may be used for the treatment of acute or chronic alcohol intoxication, for example, Can be transmitted first.

In particular aspects, the invention provides a method for silencing ALDH gene expression in a mammal (e.g., a human) in need of silencing of ALDH gene expression, said method comprising administering to the mammal one or more of the Comprising administering a therapeutically effective amount of a nucleic acid-lipid particle (e. G., A SNALP preparation) comprising an interfering RNA (e. G., SiRNA) (e.g., siRNA targeting at least one ALDH gene). In some embodiments, the administration of the nucleic acid-lipid particles comprising one or more ALDH interfering RNAs can reduce the level of ALDH RNA to a level of ALDH RNA detected in the absence of the interfering RNA (e. G., A buffer control or non-associated non-ALDH targeted interfering RNA 50%, 55%, 60%, 65%, 70%, 75%, 30%, 35%, 40%, 45%, 50% , 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 100% (or any range therein). In other embodiments, the administration of the nucleic acid-lipid particles comprising one or more ALDH-targeted interfering RNAs results in at least about 1, 2, 3, 4, or 5 times more potentiation compared to a negative control, such as a buffer control or non- , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, , 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 days or more (or any range therein).

In another aspect, the invention provides a method for silencing ALDH gene expression in a mammal (e. G., A human) in need of silencing of ALDH gene expression, said method comprising administering to the mammal one or more of the Comprising administering a therapeutically effective amount of nucleic acid-lipid particles (e. G., SNALP agents) comprising an interfering RNA (e. G., SiRNA) (e. G. SiRNA targeting at least one region of the ALDH gene) . In some embodiments, the administration of the nucleic acid-lipid particles comprising one or more ALDH interfering RNAs can reduce the level of ALDH mRNA to a level of ALDH mRNA detected in the absence of the interfering RNA (e. G., A buffer control or non-associated non-ALDH targeted interfering RNA 50%, 55%, 60%, 65%, 70%, 75%, 30%, 35%, 40%, 45%, 50% , 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 100% (or any range therein). In other embodiments, the administration of the nucleic acid-lipid particles comprising one or more ALDH-targeted interfering RNAs reduces the level of ALDH mRNA by at least about 1, 2 or 3 compared to a negative control, such as a buffer control or an unrelated non- , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, , 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 days or more (or any range therein).

In another aspect, the invention provides a method of treating and / or preventing one or more symptoms associated with alcoholism in a mammal (e.g., a human) in need of such treatment, and / or reducing the risk or likelihood of onset and / (E.g., reducing the susceptibility to and / or reducing the susceptibility thereto), delaying the expression thereof, and / or ameliorating the same, which method comprises administering to the mammal an amount of a compound of the present invention that targets ALDH gene expression Comprising administering a therapeutically effective amount of nucleic acid-lipid particles (e. G., A SNALP preparation) comprising one or more interfering RNA molecules (e.

In a further aspect, the invention provides a method of inactivating ALDH in a mammal (e. G., Human) (e. G., A human suffering from alcoholism) in need of an inactivation of ALDH, The invention includes administering to the animal a therapeutically effective amount of nucleic acid-lipid particles (e. G., SNALP agents) comprising one or more interfering RNAs (e. G., SiRNAs) described herein that target ALDH gene expression. In some embodiments, the administration of the nucleic acid-lipid particles comprising one or more ALDH-targeted interfering RNAs modulates the level of ALDH enzyme to the level of ALDH enzyme detected in the absence of the interfering RNA (e. G., A buffer control or non- At least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% 97%, 98%, 99%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , Or 100% (or any range therein).

As an example, ALDH2 mRNA is branched DNA test; can be measured by using a (quantization tijen (QuantiGene) Bahia ^® matrix (Affymetrix)). A branched DNA assay is a sandwich nucleic acid hybridization method that utilizes bDNA molecules to amplify signals from the captured target RNA. As another example, ALDH enzymatic activity can be measured spectrophotometrically by adding NAD and a substrate (e.g., acetaldehyde) to a protein sample and measuring the formation of NADH at 340 nm (see, for example, [Arolfo et al Alcoholism: Clinical and Experimental Research 2009 33 (11): 1935]; [Garver et al., Alcohol & Alcoholism 2000 35 (5): 435).

IV . Therapeutic nucleic acid

The term "nucleic acid" includes any oligonucleotide or polynucleotide, wherein a fragment containing up to 60 nucleotides is generally referred to as an oligonucleotide, and a longer fragment is referred to as a polynucleotide. In certain embodiments, the oligonucleotides of the invention are from about 15 to about 60 nucleotides in length. In some embodiments, the nucleic acid is associated with a carrier system, such as the lipid particles described herein. In certain embodiments, the nucleic acid is fully encapsulated within the lipid particle. The nucleic acid may be administered alone in the lipid particles of the present invention or may be administered (e.g., co-administered) with a peptide, polypeptide or small molecule, for example, a lipid particle comprising a conventional drug .

In the context of the present invention, the terms "polynucleotide" and "oligonucleotide" refer to polymers or oligomers of nucleotides or nucleoside monomers consisting of naturally occurring bases, sugars and meso (backbone) linking groups. The terms "polynucleotide" and "oligonucleotide" also include polymers or oligomers that contain non-naturally occurring monomers, or portions thereof, that similarly function. Such modified or substituted oligonucleotides are often desirable compared to native forms, for example due to properties such as enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nuclease.

Oligonucleotides are generally classified as deoxyribo oligonucleotides or ribo oligonucleotides. The deoxyribo oligonucleotide consists of a 5-carbon sugar, called deoxyribose, which is covalently linked to the phosphate at the 5 ' and 3 ' carbons of the sugar to form an alternating, non-branched polymer. The ribo oligonucleotide consists of a similar repeating structure in which the 5-carbon sugar is a ribose.

The nucleic acid may be single stranded DNA or RNA, or double stranded DNA or RNA, or a DNA-RNA hybrid. In a preferred embodiment, the nucleic acid is double stranded RNA. Examples of double stranded RNAs are described herein and include, for example, siRNA and other RNAi materials such as dicer-substrate dsRNA, shRNA, aiRNA, and pre-miRNA. In another embodiment, the nucleic acid is single stranded. Single-stranded nucleic acids include, for example, antisense oligonucleotides, ribozymes, mature miRNAs, and tris-forming oligonucleotides.

The nucleic acid of the present invention may generally be of various lengths depending on the particular type of nucleic acid. For example, in certain embodiments, the plasmid or gene may be from about 1,000 to about 100,000 nucleotide residues in length. In certain embodiments, oligonucleotides can range in length from about 10 to about 100 nucleotides. In various related embodiments, the single stranded, double stranded and triple stranded oligonucleotides all comprise from about 10 to about 60 nucleotides, from about 15 to about 60 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides , Or from about 20 to about 30 nucleotides in length.

In certain embodiments, the oligonucleotides (or strands thereof) of the invention are specifically hybridizing to or complementary to a target polynucleotide sequence. The terms "specifically hybridizable" and "complementarity" when used herein refer to a sufficient degree of complementarity so that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide. It is understood that the oligonucleotide need not be 100% complementary to its target nucleic acid sequence in order to be specifically hybridizable. In a preferred embodiment, the oligonucleotide is specifically hybridizable when the binding of the oligonucleotide to the target sequence inhibits the normal function of the target sequence so as to cause utility or loss of expression therefrom, and under conditions that require specific binding , That is, to a degree sufficient to avoid non-specific binding of the oligonucleotide to the non-target sequence under conditions in which assays are performed under physiological conditions in the case of in vivo assays or therapeutic treatments, or in the case of in vitro assays Lt; / RTI > Thus, the oligonucleotide may comprise one, two, three or more base substitutions relative to the region of the gene or mRNA sequence to which it is targeted or specifically hybridizes.

A. siRNA

Unmodified and modified siRNA molecules of the invention can silence ALDH gene expression. Each strand of the siRNA duplex is usually about 15 to about 60 nucleotides in length, preferably about 15 to about 30 nucleotides in length. In certain embodiments, the siRNA comprises at least one modified nucleotide. Modified siRNAs generally have less immunostimulatory activity than the corresponding unmodified siRNA sequence and retain RNAi activity against the target gene of interest. In some embodiments, the modified siRNA comprises at least one 2'OMe purine or a pyrimidine nucleotide such as 2'OMe-guanosine, 2'OMe-uridine, 2'OMe-adenosine, and / or 2'OMe-cytosine Lt; / RTI > nucleotides. Modified nucleotides can be present either within a single strand (i.e., sense or antisense) of the siRNA or within both strands. In some preferred embodiments, one or more uridine and / or guanosine nucleotides are modified (e.g., 2'OMe-modified) within one strand (i.e., sense or antisense) of siRNA or within both strands . In these embodiments, the modified siRNA further comprises one or more modified (e. G., 2'OMe-modified) adenosine and / or modified (e. G., 2'OMe-modified) cytosine nucleotides . In another preferred embodiment, only uridine and / or guanosine nucleotides are modified (e.g., 2'OMe-modified) in one strand (i.e., sense or antisense) of siRNA or both strands. The siRNA sequence may be 3 ' or 3 ' as described in Overlap (e.g., Elbashir et al., Genes Dev., 15: 188 (2001) or Nykanen et al., Cell, 107: 309 5 'overhang), or there may be no overhang (i.e., it may have a blunt end).

In certain embodiments, the selective inclusion of nucleotides modified into the double-stranded region of one or both strands of siRNA, such as 2'OMe uridine and / or guanine nucleotides, reduce or completely eliminate the immune response to the siRNA molecule do. In certain instances, the immunostimulatory properties of specific siRNA sequences and their ability to silence gene expression can be balanced or optimized by introducing a minimal and selective 2'OMe variant within the double-stranded region of siRNA duplexes . This can be achieved at therapeutically feasible siRNA doses without cytokine induction, toxicity, and off-target effects associated with the use of unmodified siRNA.

Modified siRNAs generally contain from about 1% to about 100% (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% , 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23% %, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% 90%, 95%, or 100%) modified nucleotides. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in the double stranded region of the siRNA comprise modified nucleotides. In certain other embodiments, some or all of the modified nucleotides in the double stranded region of the siRNA are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides spaced from one another. In one preferred embodiment, the modified nucleotides in the double stranded region of the siRNA are not adjacent to each other (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, There are 9 or 10 unmodified nucleotide gaps).

In some embodiments, less than about 50% (e.g., about 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41% , 39%, 38%, 37%, or 36%, preferably less than about 35%, 34%, 33%, 32%, 31%, or 30% 'OMe) nucleotides. In one aspect of these embodiments, less than about 50% of the uridine and / or guanosine nucleotides in the double stranded region of one or both strands of the siRNA are selectively (e.g., uniquely) modified. In yet another aspect of these embodiments, less than about 50% of the nucleotides in the double stranded region of the siRNA comprise a 2'OMe nucleotide, wherein the siRNA comprises 2'OMe nucleotides in both strands of the siRNA, A 2'OMe-guanosine nucleotide and at least one 2'OMe-uridine nucleotide, wherein the 2'OMe-guanosine nucleotide and the 2'OMe-uridine nucleotide comprise only the 2'OMe-uridine nucleotides present in the double- Nucleotides. In yet another aspect of these embodiments, less than about 50% of the nucleotides in the double-stranded region of the siRNA comprise a 2'OMe nucleotide, wherein the siRNA comprises 2'OMe nucleotides in both strands of the modified siRNA, Wherein the siRNA comprises a 2'OMe nucleotide selected from the group consisting of 2'OMe-guanine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, and mixtures thereof, wherein the siRNA comprises 2'OMe - Does not contain cytosine nucleotides. In a further aspect of these embodiments, less than about 50% of the nucleotides in the double stranded region of the siRNA comprise a 2'OMe nucleotide, wherein the siRNA comprises 2'OMe nucleotides in both strands of the siRNA, One 2'OMe-guanine nucleotide and at least one 2'OMe-uridine nucleotide, and the siRNA does not contain a 2'OMe-cytosine nucleotide in the double-stranded region. In yet another aspect of these embodiments, less than about 50% of the nucleotides in the double-stranded region of the siRNA comprise a 2'OMe nucleotide, wherein the siRNA comprises 2'OMe nucleotides in both strands of the modified siRNA, Comprises 2'OMe nucleotides selected from the group consisting of 2'OMe-guanine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, and mixtures thereof, and the 2'OMe nucleotides in the double- They are not adjacent to each other.

In another embodiment, about 1% to about 50% (e.g., about 5% -50%, 10% -50%, 15% -50%, 20% -50%, 25% -50%, 30% -50%, 35% -50%, 40% -50%, 45% -50%, 5% -45%, 10% -45%, 15% -45%, 20% -45 %, 25% -45%, 30% -45%, 35% -45%, 40% -45%, 5% -40%, 10% -40%, 15% -40% 25% -40%, 25% -39%, 25% -38%, 25% -37%, 25% -36%, 26% -39%, 26% -38%, 26% -37% -36%, 27% -39%, 27% -38%, 27% -37%, 27% -36%, 28% -39%, 28% -38%, 28% -37%, 28% -36% 29% -39%, 29% -38%, 29% -37%, 29% -36%, 30% -40%, 30% -39%, 30% -38%, 30% -37% 31% -37%, 31% -36%, 32% -39%, 32% -38%, 32% -37%, 32% -36%, 33% -39%, 33% -38%, 33% -37%, 33% -36%, 34% -39%, 34% -38%, 34% -37% %, 35% -40%, 5% -35%, 10% -35%, 15% -35%, 20% -35%, 21% -35%, 22% -35%, 23% -35% 35%, 29% -35%, 30% -35%, 31% -35%, 32% -35%, 25% -35%, 26% -35%, 27% -35%, 28% -35%, 33% -35%, 34% -35%, 30% -34%, 31% -34%, 32% -34%, 33% -34%, 30% -33%, 31% -33 %, 32% -33%, 30% -32%, 31% -32%, 25% -34%, 25% -33%, 25% -32%, 25% -31%, 26% -34% 26% -33%, 26% -32%, 26% -31%, 27% -34% , 27% -33%, 27% -32%, 27% -31%, 28% -34%, 28% -33%, 28% -32%, 28% -31%, 29% -34% % -33%, 29% -32%, 29% -31%, 5% -30%, 10% -30%, 15% -30%, 20% -34%, 20% -33% 30%, 20% -30%, 20% -30%, 21% -30%, 22% -30%, 23% -30%, 24% -30%, 25% -30%, 25% , 25% -28%, 25% -27%, 25% -26%, 26% -30%, 26-29%, 26-28%, 26-27%, 27-30% % -29%, 27% -28%, 28% -30%, 28% -29%, 29% -30%, 5% -25%, 10% -25%, 15% -25%, 20% 20%, 20% -27%, 20% -26%, 20% -25%, 5% -20%, 10% -20%, 15% -20%, 5% -15% , 10% -15%, or 5% -10%) of nucleotides comprise modified nucleotides. In one aspect of these embodiments, from about 1% to about 50% of the uridine and / or guanosine nucleotides in the double stranded region of one or both strands of the siRNA are selectively (e.g., uniquely) modified . In yet another aspect of these embodiments, about 1% to about 50% of the nucleotides in the double stranded region of the siRNA comprise 2'OMe nucleotides, wherein the siRNA comprises 2'OMe nucleotides in both strands of the siRNA, the siRNA comprises at least one 2'OMe-guanine nucleotide and at least one 2'OMe-uridine nucleotide, wherein the 2'OMe-guanosine nucleotide and the 2'OMe-uridine nucleotide comprise the unique 2 'Ome nucleotide. In yet another aspect of these embodiments, about 1% to about 50% of the nucleotides in the double stranded region of the siRNA comprise 2'OMe nucleotides, wherein the siRNA comprises 2'OMe nucleotides in both strands of the modified siRNA Wherein the siRNA comprises a 2'OMe nucleotide selected from the group consisting of 2'OMe-guanine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, and mixtures thereof, wherein the siRNA comprises 2'OMe-cytosine nucleotides. In a further aspect of these embodiments, about 1% to about 50% of the nucleotides in the double stranded region of the siRNA comprise 2'OMe nucleotides, wherein the siRNA comprises 2'OMe nucleotides in both strands of the siRNA, The siRNA comprises at least one 2'OMe-guanine nucleotide and at least one 2'OMe-uridine nucleotide, and the siRNA does not contain a 2'OMe-cytosine nucleotide in the double stranded region. In yet another aspect of these embodiments, about 1% to about 50% of the nucleotides in the double stranded region of the siRNA comprise 2'OMe nucleotides, wherein the siRNA comprises 2'OMe nucleotides in both strands of the modified siRNA Wherein the siRNA comprises a 2'OMe nucleotide selected from the group consisting of 2'OMe-guanine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, and mixtures thereof, wherein the 2'OMe nucleotides in the double- OMe nucleotides are not adjacent to each other.

Additional ranges, ratios, and patterns of modifications that can be introduced into siRNAs are described in U.S. Patent Publication No. 20070135372, the full text of which is incorporated herein by reference in its entirety.

One. siRNA  Selection of sequence

Suitable siRNA sequences can be identified using any means known in the art. Generally, the method described in [Elbashir et al., Nature, 411: 494-498 (2001)] and [Elbashir et al., EMBO J., 20: 6877-6888 , Nature Biotech., 22 (3): 326-330 (2004)].

As a non-limiting example, the 3 'nucleotide sequence of the AUG start codon of a transcript from a target gene of interest may comprise a nucleotide sequence (e.g. AA, NA, CC, GG, or UU, where N = C, G , Or U) (see, for example, Elbashir et al., EMBO J., 20: 6877-6888 (2001)). Nucleotides located immediately 3 'of the dinucleotide sequence are identified as potential siRNA sequences (i.e., target sequence or sense strand sequence). Generally, 19, 21, 23, 25, 27, 29, 31, 33, 35 or more nucleotides directly located 3 'of the dinucleotide sequence are identified as potential siRNA sequences. In some embodiments, the dinucleotide sequence is an AA or NA sequence and the 19 nucleotides directly located 3 ' of the AA or NA dinucleotide are identified as potential siRNA sequences. siRNA sequences are usually spaced at different positions along the length of the target gene. To further enhance the silencing efficiency of the siRNA sequence, the potential siRNA sequences may be analyzed, for example, to identify sites that do not contain regions homologous to other coding sequences in the target cell or organism. For example, a suitable siRNA sequence of about 21 base pairs will generally not have more than 16-17 consecutive base pairs homologous to the coding sequence in the target cell or organism. When the siRNA sequence is expressed from the RNA Pol III promoter, more than four consecutive A or T-deficient siRNA sequences are selected.

Once a potential siRNA sequence is identified, a complementarity sequence (i.e., an antisense strand sequence) can be designed. In addition, potential siRNA sequences can be analyzed using a variety of criteria known in the art. For example, to improve their silencing efficiency, siRNA sequences can be analyzed by a rational design algorithm to identify sequences with one or more of the following characteristics: (1) G / C content: from about 25% to about 60% G / C; (2) at least three A / Us of positions 15-19 of the sense strand; (3) absence of internal repeats; (4) A of position 19 of the sense strand; (5) A of position 3 of the sense strand; (6) the sense strand position U of U; (7) G / C member at the position 19 of the sense strand; And (8) the absence of G at position 13 of the sense strand. An siRNA design tool that includes algorithms for assigning appropriate values of each of these features and useful for siRNA selection can be found, for example, in http://ihome.ust.hk/~bokcmho/siRNA/siRNA.html. Those skilled in the art will appreciate that sequences having one or more of the above characteristics may be selected for further analysis and testing as potential siRNA sequences.

In addition, potential siRNA sequences with one or more of the following criteria may often be excluded from siRNA: (1) a sequence comprising a stretch of four or more identical bases consecutively; (2) a sequence comprising a homopolymer of G (i. E., By reducing possible non-specific effects due to structural features of these polymers); (3) a sequence comprising a triple base motif (e.g., GGG, CCC, AAA, or TTT); (4) a sequence comprising consecutively seven or more stretches of G / C; And (5) a sequence comprising direct repeats of four or more bases in the candidate, creating an internal fold-back structure. However, one of ordinary skill in the art will appreciate that sequences having one or more of the above characteristics may be selected for further analysis and testing as potential siRNA sequences.

In some embodiments, potential siRNA sequences are described, for example, in Khvorova et al., Cell, 115: 209-216 (2003); And Schwarz et al., Cell, 115: 199-208 (2003), which are incorporated herein by reference in their entirety. In another embodiment, potential siRNA sequences are described, for example, in Luo et al., Biophys. Res. Commun., 318: 303-310 (2004), which is herein incorporated by reference in its entirety. For example, the secondary structure of the target site can be modified using the Mfold algorithm (http: // mfold) to select the siRNA sequence that is favorable for use at the target site with fewer secondary structures in the form of base pairing and stem- .burnet.edu.au / rna_form). < / RTI >

Once a potential siRNA sequence has been identified, the sequence can be analyzed for the presence of any immunostimulatory property, e. G. Using an in vitro cytokine assay or an in vivo animal model. Also included are motifs within the sense and / or antisense strand of the siRNA sequence, such as 5'-GU-3 ', 5'-UGU-3', 5'-GUGU- -UGUGU-3 ", etc.) may provide an indication as to whether the sequence may be immunostimulatory. Once the siRNA molecule is found to be immunostimulatory, it can then be modified to reduce its immunostimulatory properties as described herein. As a non-limiting example, a siRNA sequence may be contacted with a mammalian responder cell under conditions that allow the cell to produce a detectable immune response to determine whether the siRNA is an immunostimulatory or non-immunostimulatory siRNA. The mammalian responder cell may be a cell of a naive mammal (i. E., A mammal not previously contacted with the gene product of the siRNA sequence). The mammalian responder cell may be, for example, peripheral blood mononuclear cells (PBMC), macrophages, and the like. The detectable immune response may include the production of cytokines or growth factors such as TNF- alpha, IFN- alpha, IFN- beta, IFN- y, IL-6, IL-12, or a combination thereof. The siRNA molecule identified as immunostimulant may then be modified to reduce its immunostimulatory properties by replacing at least one nucleotide on the sense and / or antisense strand with a modified nucleotide. For example, nucleotides having less than about 30% (e.g., about 30%, 25%, 20%, 15%, 10%, or 5%) nucleotides in the duplex region of the siRNA duplex are modified nucleotides 2'OMe nucleotides. The modified siRNA can then be contacted with the mammalian responder cell as described above to confirm that its immunostimulatory properties have been reduced or eliminated.

A suitable in vitro assay for detecting an immune response is the double monoclonal antibody sandwich immunoassay technique of David et al. (U.S. Patent No. 4,376,110); Monoclonal-polyclonal antibody sandwich assays (Wide et al., Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh (1970)); The "Western blot" method of Gordon et al. (US Patent 4,452,901); Immunoprecipitation of labeled ligands (Brown et al., J. Biol. Chem., 255: 4980-4983 (1980)); See, e.g., Raines et al., J. Biol. Chem., 257: 5154-5160 (1982); Immunocytochemistry techniques including the use of fluorophore (Brooks et al., Clin. Exp. Immunol, 39: 477 (1980)); And neutralization of activity (Bowen-Pope et al., Proc. Natl. Acad. Sci. USA, 81: 2396-2400 (1984)). In addition to the immunoassays described above, U.S. Patent 3,817,827; 3,850,752; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; And 4,098,876 are available. The disclosure of which is incorporated herein by reference in its entirety for all purposes.

Non-limiting examples of in vivo models for detecting immune responses are described, for example, in Judge et al., Mol. Ther., 13: 494-505 (2006). In certain embodiments, the assay can be performed as follows: (1) the siRNA can be administered by standard intravenous injection in the lateral tail vein; (2) blood can be collected by cardiac puncture about 6 hours after administration and treated as plasma for cytokine analysis; (3) Cytokines can be quantitated using a sandwich ELISA kit according to the manufacturer's instructions (e.g., mouse and human IFN-alpha (PBL Biomedical; Piscataway, NJ); human (BD Biosciences; San Diego, Calif., USA) and IL-6 and TNF-alpha (eBioscience; San Diego, ).

Monoclonal antibodies that specifically bind to cytokines and growth factors are commercially available from a variety of sources and can be generated using methods known in the art (see, for example, Kohler et al., Nature, 256: 495-497 (1975) and Harlow and Lane, ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publication, New York (1999)). The production of monoclonal antibodies has been previously described in the literature and can be carried out by any means known in the art (Buhring et al., Hybridoma, Vol. 10, No. 1, pp. 77-78 (1991)). In some methods, monoclonal antibodies are labeled (e. G., Using any composition detectable by spectroscopic, photochemical, biochemical, electrical, optical, or chemical means) to facilitate detection.

2. siRNA  Generation of molecules

siRNAs may be provided in a variety of forms including, for example, one or more isolated small interfering RNA (siRNA) duplexes, longer double-stranded RNAs (dsRNAs), or siRNAs or dsRNAs transcribed from a transcription cassette in a DNA plasmid. In some embodiments, siRNAs can be produced by enzymes or by partial / total organic synthesis, and modified ribonucleotides can be introduced by in vitro enzymatic or organic synthesis. In certain cases, each strand is chemically prepared. Methods for the synthesis of RNA molecules are well known in the art and are, for example, chemical synthesis methods as described or described herein in Verma and Eckstein (1998).

The RNA population may be used to provide long precursor RNAs or long precursor RNAs with substantial or complete identity to the selected target sequence may be used to produce the siRNA. The RNA may be isolated and / or synthesized and / or cloned according to methods well known to those skilled in the art. The RNA may be a mixed population (obtained from a cell or tissue, transcribed, subtracted, or selected from cDNA, etc.) or may represent a single target sequence. The RNA may be naturally occurring (e.g., isolated from a tissue or cell sample), synthesized in vitro (e.g., using T7 or SP6 polymerase and PCR products or cloned cDNA), or chemically synthesized .

In synthetic RNA, complement to form long dsRNA is also transcribed in vitro and hybridized to form dsRNA. If a naturally occurring population of RNAs is used, the RNA complement is also provided, for example, by transcribing the cDNA corresponding to the RNA population or using an RNA polymerase (e. G., Digestion with E. coli RNAse III or Dicer To form the dsRNA for. The precursor RNA is then hybridized to form double stranded RNA for digestion. The dsRNA can be administered directly to the subject or can be digested in vitro prior to administration.

Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, preparing and screening cDNA libraries, and performing PCR are described in PCR methods (U.S. Patent Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (See, for example, Gubler and Hoffman, Gene, 25: 263-269 (1983); Sambrook et al., Supra), as described in Innis et al., Eds ; Ausubel et al., Supra). Expression libraries are also known to those skilled in the art. Additional basic texts disclosing the general methods of use in the present invention are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); [Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990)]; And Current Protocols in Molecular Biology (Ausubel et al., Eds., 1994). The disclosure of which is incorporated herein by reference in its entirety for all purposes.

Preferably, the siRNA is chemically synthesized. Oligonucleotides comprising the siRNA molecules of the invention are described in Usman et al., J. Am. Chem. Soc., 109: 7845 (1987); [Scaringe et al., Nucl. Acids Res., 18: 5433 (1990); [Wincott et al., Nucl. Acids Res., 23: 2677-2684 (1995); And Wincott et al., Methods Mol. Bio., 74: 599 (1997)). ≪ / RTI > Synthesis of oligonucleotides utilizes conventional nucleic acid protection and coupling groups such as 5'-terminal dimethoxytrityl and 3'-terminal phosphoramidite. As a non-limiting example, small scale synthesis can be performed in an Applied Biosystems synthesizer using a 0.2 μmol scale protocol. Alternatively, synthesis at 0.2 μmol scale can be performed in a 96-well plate synthesizer from Protogene (Palo Alto, Calif., USA). However, larger or smaller scale syntheses are also included within the scope of the present invention. Suitable reagents, RNA deprotection methods, and RNA purification methods for oligonucleotide synthesis are known to those skilled in the art.

siRNA molecules can also be synthesized via tandem synthesis techniques where both strands are synthesized as a single continuous oligonucleotide fragment or strand separated by a cleavable linker and then cleaved into separate fragments or strands , Which hybridize to form siRNA duplexes. The linker may be a polynucleotide linker or a non-nucleotide linker. The tandem synthesis of siRNAs can be easily tailored for both multiple well / multiple plate synthesis platforms and large scale synthesis platforms using batch reactors, synthesis columns, and the like. Alternatively, the siRNA molecule can be assembled from two distinct oligonucleotides, wherein one oligonucleotide comprises the sense strand of the siRNA and the other comprises the antisense strand. For example, each strand may be synthesized separately and linked together by hybridization or ligation after synthesis and / or deprotection. In certain other examples, siRNA molecules can be synthesized as a single continuous oligonucleotide fragment, wherein the self-complementary sense and antisense regions are hybridized to form siRNA duplexes with a hairpin secondary structure.

3. siRNA  Variation of sequence

In certain aspects, the siRNA molecule comprises two strands, and a duplex with at least one modified nucleotide in the double stranded region, wherein each strand is about 15 to about 60 nucleotides in length. Advantageously, the modified siRNA is less immunostimulatory than the corresponding unmodified siRNA sequence, but retains the ability to silence the expression of the target sequence. In a preferred embodiment, the degree of chemical modification introduced into the siRNA molecule affects the balance between reducing or eliminating the immunostimulatory properties of the siRNA and retention of RNAi activity. As a non-limiting example, a siRNA molecule targeting a gene of interest may contain a selective uridine and / or guanosine in the siRNA duplex to eliminate the immune response produced by the siRNA while retaining its ability to silence target gene expression (E. G., Less than about 30%, 25%, 20%, 15%, 10%, or 5% variants) at the new nucleotide.

Examples of modified nucleotides suitable for use in the present invention are 2'-O-methyl (2'OMe), 2'-deoxy-2'-fluoro (2'F), 2'- -Methyl, 2'-O- (2-methoxyethyl) (MOE), 4'-thio, 2'-amino, or 2'-C-allyl group. Also, for example, Saenger, Principles of Nucleic Acid Structure, Springer-Verlag Ed. (1984)] are also suitable for use in siRNA molecules. The modified nucleotides include, but are not limited to, a locked nucleic acid (LNA) nucleotide (eg, 2'-O, 4'-C-methylene- (D-ribofuranosyl) 2'-deoxy-2'-fluoro (2'F) nucleotide, 2'-deoxy-2'-chloro (2'Cl) nucleotides, and 2'-azido nucleotides. In certain instances, the siRNA molecule described herein comprises one or more G-clamp nucleotides. G-clamp nucleotides refer to modified cytosine analogs that give modifications the ability to hydrogen bond to both Watson-Crick and Hoogsteen faces of complementary guanine nucleotides in the duplex (see, e. For example, Lin et al., J. Am. Chem. Soc., 120: 8531-8532 (1998)). In addition, nucleotide base analogues such as C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azolescarboxamide, and nitrazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole , And nucleotides having 6-nitroindole (see, for example, Loakes, Nucl. Acids Res., 29: 2437-2447 (2001)) can be introduced into siRNA molecules.

In certain embodiments, the siRNA molecule may further comprise one or more chemical modifications, such as a cap cap moiety, a phosphate backbone modification, and the like. Examples of terminal cap moieties include, but are not limited to, inverted deoxy abasic residues, glyceryl modifications, 4 ', 5'-methylene nucleotides, 1- (β-D-erythropyranosyl) , 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotide, a-nucleotide, modified nucleotide nucleotide, ', 4'-seco nucleotide, bicyclic 3,4-dihydroxybutyl nucleotide, bicyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety, 3'- 3'-2'-inverted basic moiety, 3'-2'-inverted basic moiety, 5'-5'-inverted nucleotide moiety, 5'- A 5'-reversed anhydrous basic moiety, a 3'-5'-reversed deoxybased basic moiety, a 5'-amino-al Aminodecylphosphate, 1,2-amidodecylphosphate, hydroxypropylphosphate, 1,4-butanediolphosphate, 3'-diamino-2-propylphosphate, Phosphoramidate, 5'-phosphoramidate, hexylphosphate, aminohexylphosphate, 3'-phosphate, 5'-amino, 3'-phosphorothioate, 5'-phosphorothioate, And crosslinked or non-crosslinked methyl phosphonates or 5'-mercapto moieties (see, for example, U.S. Patent 5,998,203; Beaucage et al., Tetrahedron 49: 1925 (1993)). Non-limiting examples of phosphate backbone modifications (i. E., Generating modified internucleotide linkages) include, but are not limited to, phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, (E.g., Hunziker et al., Nucleic Acid Analogues < (R) >), (See, for example, Mesmaeker et al., Novel Backbone Replacement for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39 (1994)). . The chemical modification may occur at the 5'-end and / or the 3'-end of the sense strand, antisense strand, or both strands of the siRNA. These references are incorporated herein by reference in their entirety for all purposes.

In some embodiments, the sense and / or antisense strand of the siRNA molecule comprises about 1 to about 4 (e.g., 1, 2, 3 or 4) 2'-deoxyribonucleotides, modified Terminal overhang with a 2'OMe) and / or unmodified uridine ribonucleotide, and / or any other combination of modified (e.g., 2'OMe) and unmodified nucleotides As shown in FIG.

Additional examples of variants of the nucleotides and the types of chemical modifications that can be introduced into the siRNA molecule are described, for example, in UK Patent GB 2,397,818 B and U.S. Patents 20040192626, 20050282188, and 20050282188, which are incorporated herein by reference in their entirety for all purposes 20070135372.

The siRNA molecules described herein may optionally comprise one or more non-nucleotides in one or both strands of the siRNA. As used herein, the term "non-nucleotide" refers to any group or compound that can be incorporated into a nucleic acid strand in place of one or more nucleotide units, including sugar and / or phosphate substitutions, . Group or compound is conventionally non-basic in that it does not contain a recognized nucleotide base, such as adenosine, guanine, cytosine, uracil, or thymine, and thus lacks a base at the 1'-position.

In another embodiment, the chemical modification of the siRNA comprises attaching the conjugate to the siRNA molecule. The conjugate may be attached at the 5 ' and / or 3 ' -end of the sense and / or antisense strand of the siRNA via a covalent attachment, e.g., biodegradable linker. The conjugate can also be attached to the siRNA via, for example, a carbamate group or other linker (see, for example, U.S. Patent Nos. 20050074771, 20050043219, and 20050158727). In certain cases, the conjugate is a molecule that facilitates delivery of the siRNA into the cell. Examples of suitable conjugate molecules for attachment to siRNA include but are not limited to steroids such as cholesterol, glycols such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folate (Such as folic acid, folate analogs and derivatives thereof), sugars (such as galactose, galactosamine, N-acetylgalactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, (For example, see U.S. Patent Nos. 20030130186, 20040110296, and 20040249178; U.S. Patent 6,753,423). Other examples include lipophilic moieties, vitamins, polymers, peptides, proteins, nucleic acids, small molecules, oligosaccharides, carbohydrate clusters, intercalators, minor groove binders, Cleavage agents, and cross-linker conjugate molecules. Other examples are 2'-O-alkylamines, 2'-O-alkoxyalkylamines, polyamines, C5-cationic modified pyrimidines, cationic peptides, guanidinium groups, and amidinium groups described in US Patent Publication 20050153337 , And cationic amino acid conjugate molecules. Additional examples include hydrophobic groups, membrane active compounds, cytopathic compounds, cell targeting signals, interaction modifiers, and steric stabilizer conjugate molecules described in US Patent Publication 20040167090. A further example includes the conjugate molecule described in U. S. Patent Publication 20050239739. < RTI ID = 0.0 > The type of conjugate used and the degree of conjugation to the siRNA molecule can be assessed for improved pharmacokinetic profile, bioavailability, and / or stability of siRNA while retaining RNAi activity. Thus, one skilled in the art can use any of a variety of well-known in vitro cell cultures or in vivo animal models to screen for siRNA molecules with various conjugates attached thereto to confirm that they have improved properties and complete RNAi activity. The disclosure of which is incorporated herein by reference in its entirety for all purposes.

4. Exemplary siRNA  Embodiment

In some embodiments, each strand of the siRNA molecule has a length of about 15 to about 60 nucleotides (e.g., about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 Nucleotide length, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). In one particular embodiment, the siRNA is chemically synthesized. The siRNA molecules of the invention can silence the expression of a target sequence in vitro and / or in vivo.

In another embodiment, the siRNA comprises at least one modified nucleotide. In certain embodiments, the siRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleotides in the double stranded region. In certain embodiments, less than about 50% (e.g., about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5 %) Of nucleotides comprise modified nucleotides. In a preferred embodiment, about 1% to about 50% (e.g., about 5% -50%, 10% -50%, 15% -50%, 20% -50%, 25% -50%, 30% -50%, 35% -50%, 40% -50%, 45% -50%, 5% -45%, 10% -45%, 15% -45%, 20% -45 %, 25% -45%, 30% -45%, 35% -45%, 40% -45%, 5% -40%, 10% -40%, 15% -40% 25% -40%, 30% -40%, 35% -40%, 5% -35%, 10% -35%, 15% -35%, 20% -35%, 25% -35% -35%, 5% -30%, 10% -30%, 15% -30%, 20% -30%, 25% -30%, 5% -25%, 10% -25%, 15% -25 , 20% -25%, 5% -20%, 10% -20%, 15% -20%, 5% -15%, 10% -15%, or 5% -10% Nucleotides.

In a further embodiment, the siRNA is selected from the group consisting of 2'-O-methyl (2'OMe) nucleotides, 2'-deoxy-2'-fluoro (2'F) nucleotides, 2'-deoxynucleotides, - (2-methoxyethyl) (MOE) nucleotides, lock nucleic acid (LNA) nucleotides, and mixtures thereof. In a preferred embodiment, the siRNA is a 2'OMe nucleotide (e.g., a 2'OMe purine and / or a pyrimidine nucleotide) such as a 2'OMe-guanosine nucleotide, a 2'OMe-uridine nucleotide, a 2'OMe- Adenosine nucleotides, 2'OMe-cytosine nucleotides, or mixtures thereof. In one particular embodiment, the siRNA comprises at least one 2'OMe-guanosine nucleotide, 2'OMe-uridine nucleotides, or a mixture thereof. In certain cases, the siRNA does not include 2'OMe-cytosine nucleotides. In another embodiment, the siRNA comprises a hairpin loop structure.

In certain embodiments, the siRNA comprises modified nucleotides in one strand (i.e., sense or antisense) of the double stranded region of the siRNA molecule or in both strands. Preferably, the uridine and / or guanine nucleotides are modified at selective positions within the double stranded region of the siRNA duplex. In the case of Uridine nucleotide modifications, at least 1, 2, 3, 4, 5, 6 or more uridine nucleotides in the sense and / or antisense strand are modified uridine nucleotides, such as 2'OMe-uridine nucleotides . In some embodiments, all uridine nucleotides in the sense and / or antisense strand are 2'OMe-uridine nucleotides. In a guanosine nucleotide variant, at least 1, 2, 3, 4, 5, 6 or more guanosine nucleotides in the sense and / or antisense strand may be modified guanosine nucleotides, such as 2'OMe-guanosine nucleotides have. In some embodiments, all guanosine nucleotides in the sense and / or antisense strand are 2'OMe-guanosine nucleotides.

In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7 or more 5'-GU-3 'motifs in the siRNA sequence remove the 5'-GU-3' motif For example, by introducing a mismatch and / or by introducing modified nucleotides such as 2 ' Ome nucleotides. The 5'-GU-3 'motif may be present in the sense strand, antisense strand, or both strands of the siRNA sequence. The 5'-GU-3 'motifs can be contiguous to each other or, alternatively, they can be linked by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides Can be separated.

In some embodiments, the modified siRNA molecule is less immunostimulatory than the corresponding unmodified siRNA sequence. In such embodiments, the modified siRNA molecule with reduced immunostimulatory properties advantageously retains RNAi activity against the target sequence. In another embodiment, the immunostimulatory properties of the modified siRNA molecule and its ability to silence target gene expression are determined within the siRNA sequence, e. G., Within the double stranded region of the siRNA duplex, Can be balanced or optimized by introduction. In certain instances, the modified siRNA is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% 99%, 95%, 96%, 97%, 98%, 99%, 90%, 55%, 60%, 65%, 70%, 75%, 80%, 85% , Or 100% smaller. The immunostimulatory properties of the modified siRNA molecule and the corresponding unmodified siRNA molecule can be determined, for example, by systemic administration in mammals or by transfection of mammalian responder cells using appropriate lipid-based delivery systems (e.g., the SNALP delivery system described herein) It will be readily appreciated by those skilled in the art that determination can be made by measuring levels of INF-alpha and / or IL-6 after about 2 to about 12 hours.

In other embodiments, the modified siRNA molecule has an IC ₅₀ (i.e., 1/2 of the maximum inhibitory concentration) that is less than or equal to 10 times the corresponding unmodified siRNA (i.e., the modified siRNA has a corresponding ratio less than 10 times the IC ₅₀ of a modified siRNA has an IC ₅₀ or equivalent thereof). In another embodiment, the modified siRNA has an IC ₅₀ that is less than or equal to three times the corresponding unmodified siRNA sequence. In another embodiment, the modified siRNA has an IC ₅₀ that is less than or equal to two times the corresponding unmodified siRNA. Those skilled in the art will readily recognize that dose-response curves can be generated and the IC ₅₀ values for the modified siRNA and the corresponding unmodified siRNA can be readily determined using methods known to those skilled in the art.

In another embodiment, the unmodified or modified siRNA molecule is capable of inhibiting the expression of the target sequence relative to a negative control (e.g., a buffer alone, a siRNA sequence targeting different genes, a scrambled siRNA sequence, etc.) At least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% 87%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 94%, 95%, 96%, 97%, 98%, 99%, or 100% silent.

In another embodiment, a modified siRNA molecule has at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or more of the expression of the target sequence relative to the corresponding unmodified siRNA sequence. , 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% have.

In some embodiments, the siRNA molecule does not include a phosphate backbone variant, for example, on the sense and / or antisense strand of the double stranded region. In other embodiments, the siRNA comprises, for example, 1, 2, 3, 4 or more phosphate backbone modifications within the sense and / or antisense strand of the double stranded region. In a preferred embodiment, the siRNA does not comprise a phosphate backbone variant.

In a further embodiment, the siRNA does not contain 2 ' -deoxynucleotides, for example, in the sense and / or antisense strand of the double-stranded region. In a further embodiment, the siRNA comprises, for example, 1, 2, 3, 4 or more 2'-deoxynucleotides in the sense and / or antisense strand of the double stranded region. In a preferred embodiment, the siRNA does not comprise 2 ' -deoxynucleotides.

In certain cases, the nucleotide at the 3'-end of the double-stranded region in the sense and / or antisense strand is not a modified nucleotide. In certain other examples, nucleotides (e.g., within 1, 2, 3, or 4 nucleotides at the 3'-end) near the 3'-end of the double-stranded region in the sense and / or antisense strand comprise modified nucleotides no.

The siRNA molecules described herein may have 3 'overhangs of one, two, three, four or more nucleotides on one or both sides of the double stranded region, or may have overhangs on one or both sides of the double stranded region (I. E. May have a blunt end). ≪ / RTI > In certain embodiments, the 3 ' overhang on the sense and / or antisense strand is independently 1, 2, 3, 4 or more modified nucleotides such as 2 ' Ome nucleotides and / Lt; RTI ID = 0.0 > nucleotides < / RTI >

In certain embodiments, the siRNA targeting ALDH RNA or ALDH mRNA is administered using a carrier system, such as nucleic acid-lipid particles. In a preferred embodiment, the nucleic acid-lipid particle comprises (a) one or more siRNA molecules targeting ALDH; (b) cationic lipids (e. g., DLinDMA, DLenDMA, DLin-K-C2-DMA, and / or y-DLenDMA); And (c) non-cationic lipids (e.g., DPPC, DSPC, DSPE, and / or cholesterol). In certain instances, the nucleic acid-lipid particles may additionally comprise a conjugated lipid (e.g., PEG-DAA and / or POZ-DAA) that interferes with the aggregation of the particles.

In addition to its utility in silencing the expression of any of the above-described ALDH genes for therapeutic purposes, the siRNAs described herein are also useful in research and development applications and diagnostic, prophylactic, prognostic, clinical, and other health applications. As a non-limiting example, siRNA may be used in a target identification study aimed at testing whether a particular member of the ALDH gene family has the potential to become a therapeutic target.

B. Dicer -temperament dsRNA

As used herein, the term "dicer-substrate dsRNA" or "precursor RNAi molecule" refers to any molecule that is treated in vivo by a dicer to produce an active siRNA that is incorporated into a RISC complex for RNA interference of a target gene. Are intended to include precursor molecules.

In one embodiment, the dicer-substrate dsRNA has a length sufficient to be processed by the dicer to produce siRNA. According to this embodiment, the dicer-substrate dsRNA may be (i) about 25 to about 60 nucleotides in length (e.g., about 25-60, 25-55, 25-50, 25-45, 25-40, 25 -35, or 25-30 nucleotides in length), preferably about 25 to about 30 nucleotides in length (e.g., 25, 26, 27, 28, 29, or 30 nucleotides in length) (Also referred to as sense strand), and (ii) a second oligonucleotide sequence (also referred to as antisense strand) annealing to a first sequence under biological conditions, such as those found in the cytoplasm of a cell. The second oligonucleotide sequence is about 25 to about 60 nucleotides in length (e.g., about 25-60, 25-55, 25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides Length), and preferably about 25 to about 30 nucleotides in length (e.g., 25, 26, 27, 28, 29, or 30 nucleotides in length). In addition, one of the sequences of the dicer-substrate dsRNA, particularly the region of the antisense strand, comprises at least about 19 nucleotides sufficiently complementary to the nucleotide sequence of the RNA produced from the target gene to trigger an RNAi reaction, 19 to about 60 nucleotides (e.g. about 19-60, 19-55, 19-50, 19-45, 19-40, 19-35, 19-30, or 19-25 nucleotides) Has a sequence length of about 19 to about 23 nucleotides (e.g., 19, 20, 21, 22, or 23 nucleotides).

In a second embodiment, the dicer-substrate dsRNA has some properties that enhance its processing by the dicer. According to this embodiment, the dsRNA is of sufficient length to be processed by the dicer to produce siRNA, and has at least one of the following characteristics: (i) the dsRNA is asymmetric and, for example, 'With / without overhang; (ii) the dsRNA has a modified 3'-end on the sense strand to induce orientation of the dicer binding and treatment of the dsRNA with the active siRNA. According to this latter embodiment, the sense strand comprises about 22 to about 28 nucleotides, and the antisense strand comprises about 24 to about 30 nucleotides.

In one embodiment, the dicer-substrate dsRNA has an overhang on the 3'-end of the antisense strand. In another embodiment, the sense strand is modified for treatment with a suitable modifier located at the 3'-end of the sense strand and the dicer binding. Suitable modifying agents include nucleotides such as deoxyribonucleotides, bicyclo nucleotides, and the like, and sterically hindered molecules, such as fluorescent molecules, and the like. When a nucleotide modification agent is used, they replace the ribonucleotide in the dsRNA so that the length of the dsRNA does not change. In another embodiment, the dicer-substrate dsRNA has an overhang on the 3'-end of the antisense strand and the sense strand is modified for dicer treatment. In another embodiment, the 5'-end of the sense strand has a phosphate. In another embodiment, the 5'-end of the antisense strand has a phosphate. In another embodiment, both the antisense strand or sense strand or both strands have at least one 2'-O-methyl (2'OMe) modified nucleotide. In another embodiment, the antisense strand contains a 2'OMe modified nucleotide. In another embodiment, the antisense strand comprises a 3'-overhang consisting of 2'OMe modified nucleotides. The antisense strand may also comprise additional 2'OMe modified nucleotides. Sense and antisense strands anneal under biological conditions, such as those found in the cytoplasm of a cell. In addition, one of the sequences of the dicer-substrate dsRNA, particularly the region of the antisense strand, has a sequence length of at least about 19 nucleotides, wherein these nucleotides are within the 21-nucleotide region adjacent to the 3'-end of the antisense strand, It is sufficiently complementary to the nucleotide sequence of the RNA produced from the target gene. In addition, according to this embodiment, the dicer-substrate dsRNA may also have one or more of the following additional properties: (a) the antisense strand exhibits rightward movement from a typical 21-mer (i.e., Includes the nucleotide on the right side of the molecule as compared to a typical 21-mer); (b) the strand may not be completely complementary, i.e., the strand may comprise simple mismatch pairing; (c) a base modification, such as a lock nucleic acid (s), can be included within the 5'-end of the sense strand.

In a third embodiment, the sense strand comprises about 25 to about 28 nucleotides (e.g., 25, 26, 27, or 28 nucleotides) wherein the two nucleotides on the 3'- Lt; / RTI > The sense strand contains a phosphate at the 5'-end. The antisense strand comprises about 26 to about 30 nucleotides (e.g., 26, 27, 28, 29, or 30 nucleotides) and contains 3'-overhangs of 1-4 nucleotides. Nucleotides containing 3'-overhangs are modified using 2'OMe modified ribonucleotides. The antisense strand contains alternating 2'OMe modified nucleotides starting from the first monomer of the antisense strand adjacent to the 3'-overhang and extending 15-19 nucleotides from the first monomer adjacent to the 3'-overhang . For example, when the first base at the 5'-end of the antisense strand in the 27-nucleotide antisense strand is counted as position number 1, the 2'OMe strain is the base 9,11,13,15,17,19,21, 23, 25, 26, and 27, respectively. In one embodiment, the dicer-substrate dsRNA has the following structure:

(서열 1)

(SEQ ID NO: 1)

(서열 2)

(SEQ ID NO: 2)

Here, "X" = RNA, "p" = phosphate group, "X" = 2'OMe RNA, "Y" is an overhang consisting of 1, Domain, and "D" = DNA. The upper strand is the sense strand and the lower strand is the antisense strand.

In a fourth embodiment, a dicer-substrate dsRNA has several properties that enhance its processing by the dicer. According to this embodiment, the dsRNA has a length and at least one of the following characteristics sufficient to be processed by the dicer to produce siRNA: (i) the dsRNA is asymmetric and, for example, Have overhang; (ii) the dsRNA has a modified 3'-end on the antisense strand to induce orientation of the dicer binding and treatment of the dsRNA with the active siRNA. According to this embodiment, the sense strand comprises about 24 to about 30 nucleotides (e.g., 24, 25, 26, 27, 28, 29, or 30 nucleotides) and the antisense strand comprises about 22 to about 28 (E. G., 22, 23, 24, 25, 26, 27, or 28 nucleotides). In one embodiment, the dicer-substrate dsRNA has an overhang on the 3'-end of the sense strand. In another embodiment, the antisense strand is modified for diester binding and processing by a suitable modifier located at the 3'-end of the antisense strand. Suitable modifying agents include nucleotides such as deoxyribonucleotides, bicyclo nucleotides, and the like, and sterically hindered molecules, such as fluorescent molecules, and the like. When a nucleotide modification agent is used, they replace the ribonucleotide in the dsRNA so that the length of the dsRNA does not change. In another embodiment, the dsRNA has an overhang on the 3'-end of the sense strand and the antisense strand is modified for dicer treatment. In one embodiment, the antisense strand has a 5 ' -phosphate. Sense and antisense strands anneal under biological conditions, such as those found in the cytoplasm of a cell. In addition, one of the sequences of the dsRNA, particularly the region of the antisense strand, has a sequence length of at least 19 nucleotides, wherein these nucleotides are adjacent to the 3'-end of the antisense strand and have a nucleotide sequence of RNA produced from the target gene It is sufficiently complementary. Additionally, according to this embodiment, the dicer-substrate dsRNA may also have one or more of the following additional characteristics: (a) the antisense strand exhibits a left shift from a typical 21-mer (i.e., the antisense strand Including nucleotides on the left side of the molecule as compared to a typical 21-mer); (b) the strand may not be completely complementary, i.e., the strand may comprise simple mismatch pairing.

In a preferred embodiment, the dicer-substrate dsRNA has an asymmetric structure in which the sense strand is 25-base pair length and the antisense strand is a 27-base pair length with a 3 ' overhang of two bases. In certain cases, the dsRNA having an asymmetric structure additionally contains two deoxynucleotides at the 3'-end of the sense strand instead of two ribonucleotides. In certain other examples, the dsRNA having an asymmetric structure additionally contains a 2'OMe variant at positions 9, 11, 13, 15, 17, 19, 21, 23, and 25 of the antisense strand The first base at position 5 ' -terminal is position 1). In certain further examples, the dsRNA having an asymmetric structure further comprises a 3'-overhang on the antisense strand, comprising 1, 2, 3, or 4 2'OMe nucleotides (e.g., Overhangs of the 2'OMe nucleotides at positions 26 and 27).

In another embodiment, a dicer-substrate dsRNA can be engineered by first selecting an antisense strand siRNA sequence of at least 19 nucleotides in length. In some instances, the antisense siRNA is modified to include about 5 to about 11 ribonucleotides at the 5'-end to provide a length of about 24 to about 30 nucleotides. If the length of the antisense strand is 21 nucleotides, a length of 3-9, preferably 4-7, or more preferably 6 nucleotides may be added on the 5'-end. The added ribonucleotide may be complementary to the target gene sequence, but complete complementarity between the target sequence and the antisense siRNA is not required. That is, the resulting antisense siRNA is substantially complementary to the target sequence. A sense strand having about 22 to about 28 nucleotides is then produced. The sense strand is substantially complementary to the antisense strand and therefore anneals to the antisense strand under biological conditions. In one embodiment, the sense strand is synthesized to contain a modified 3'-end to induce dicer treatment of the antisense strand. In another embodiment, the antisense strand of the dsRNA has a 3'-overhang. In a further embodiment, the sense strand is synthesized to contain a modified 3'-end for dicer binding and processing, and the antisense strand of the dsRNA has a 3'-overhang.

In a related embodiment, the antisense siRNA may be modified to include about 1 to about 9 ribonucleotides on the 5'-end to provide a length of about 22 to about 28 nucleotides. If the length of the antisense strand is 21 nucleotides, 1-7, preferably 2-5, or more preferably 4 ribonucleotides may be added on the 3'-end. The added ribonucleotides may have any sequence. The added ribonucleotide may be complementary to the target gene sequence, but complete complementarity between the target sequence and the antisense siRNA is not required. That is, the resulting antisense siRNA is substantially complementary to the target sequence. A sense strand having about 24 to about 30 nucleotides is then produced. The sense strand is substantially complementary to the antisense strand and therefore anneals to the antisense strand under biological conditions. In one embodiment, the antisense strand is synthesized to contain a modified 3'-end to induce dicer treatment. In another embodiment, the sense strand of the dsRNA has a 3 ' overhang. In a further embodiment, the antisense strand is synthesized to contain a modified 3'-end for dicer binding and processing, and the antisense strand of the dsRNA has a 3'-overhang.

Suitable dicer-substrate dsRNA sequences can be identified, synthesized, and modified using any means known in the art for designing, synthesizing, and modifying siRNA sequences. In certain embodiments, the dicer-substrate dsRNA of the invention can silence ALDH gene expression. In certain embodiments, the dicer-substrate dsRNA targeting ALDH mRNA is administered using a carrier system, such as nucleic acid-lipid particles. In a preferred embodiment, the nucleic acid-lipid particle comprises (a) one or more dicer-substrate dsRNA molecules that target ALDH gene expression; (b) cationic lipids (e. g., DLinDMA, DLenDMA, DLin-K-C2-DMA, and / or y-DLenDMA); And (c) non-cationic lipids (e.g., DPPC, DSPC, DSPE, and / or cholesterol). In certain cases, the nucleic acid-lipid particles may further comprise a conjugated lipid (e. G., PEG-DAA and / or POZ-DAA) that prevents aggregation of the particles.

Additional embodiments relating to the dicer-substrate dsRNA of the present invention and methods of designing and synthesizing such dsRNA are described in U.S. Patent Publication Nos. 20050244858, 20050277610, and 20070265220, 2011 / 0071208.

C. shRNA

"Small hairpin RNA" or "short hairpin RNA" or "shRNA" includes short RNA sequences that make tight hairpin turns that can be used to silence gene expression through RNA interference. The shRNAs of the invention can be chemically synthesized or transcribed from a transcription cassette in a DNA plasmid. The shRNA hairpin structure is cleaved into siRNA by cell machinery and then bound to RNA-induced silencing complex (RISC).

The shRNAs of the invention generally have a length of about 15-60, 15-50, or 15-40 (duplex) nucleotides, more usually about 15-30, 15-25, or 19-25 (duplex) nucleotides (Duplex) nucleotides in length (for example, each complementary sequence of a double-stranded shRNA is 15-60, 15-50, 15 20-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or 21-23 nucleotides in length, double-stranded shRNAs are about 15-60, 15- -50, 15-40, 15-30, 15-25, or 19-25 base pair lengths, preferably about 18-22, 19-20, or 19-21 base pair lengths). The shRNA duplex may comprise about 1 to about 4 nucleotides on the antisense strand or a 3 ' overhang of about 2 to about 3 nucleotides and / or a 5 ' -phosphate end on the sense strand. In some embodiments, the shRNA is from about 15 to about 60 nucleotides in length (e.g., about 15-60, 15-55, 15-50, 15-45, 15-40, 15-35, 15-25 nucleotides in length), preferably about 19 to about 40 nucleotides in length (e.g., about 19-40, 19-35, 19-30, or 19-25 nucleotides in length) The sense strand and / or antisense strand sequence of about 19 to about 23 nucleotides in length (e.g., 19, 20, 21, 22, or 23 nucleotides in length).

Non-limiting examples of shRNAs include double-stranded polynucleotide molecules assembled from a single-stranded molecule, wherein the sense and antisense regions are joined by a nucleic acid-based or non-nucleic acid-based linker; And double-stranded polynucleotide molecules having a hairpin secondary structure with self-complementary sense and antisense regions. In a preferred embodiment, the sense and antisense strands of the shRNA comprise about 1 to about 25 nucleotides, about 2 to about 20 nucleotides, about 4 to about 15 nucleotides, about 5 to about 12 nucleotides, or 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides Lt; / RTI >

Suitable shRNA sequences can be identified, synthesized, and modified using any means known in the art for designing, synthesizing, and modifying siRNA sequences. In certain embodiments, the shRNA of the invention can silence ALDH gene expression. In certain embodiments, an shRNA targeting ALDH mRNA is administered using a carrier system, such as nucleic acid-lipid particles. In a preferred embodiment, the nucleic acid-lipid particle comprises (a) one or more shRNA molecules that target ALDH gene expression; (b) cationic lipids (e. g., DLinDMA, DLenDMA, DLin-K-C2-DMA, and / or y-DLenDMA); And (c) non-cationic lipids (e.g., DPPC, DSPC, DSPE, and / or cholesterol). In certain cases, the nucleic acid-lipid particles may further comprise a conjugated lipid (e. G., PEG-DAA and / or POZ-DAA) that prevents aggregation of the particles.

Additional embodiments of shRNAs of the invention, and methods of designing and synthesizing such shRNAs, are described in U.S. Patent Application Publication No. 2011/0071208, the full text of which is incorporated herein by reference in its entirety.

D. aiRNA

As with siRNAs, asymmetric interfering RNAs (aiRNAs) mobilize RNA-induced silencing complexes (RISCs) and mediate sequence-specific cleavage of the target sequence between nucleotides 10 and 11 relative to the 5 ' end of the antisense strand Can induce effective silencing of various genes in animal cells (Sun et al., Nat. Biotech., 26: 1379-1382 (2008)). Generally, an aiRNA molecule contains short RNA duplexes with sense and antisense strands, wherein the duplexes contain overhangs at the 3 ' and 5 ' ends of the antisense strand. The aiRNA is generally asymmetric because the sense strand is shorter at both ends than the complementary antisense strand. In some aspects, the aiRNA molecules can be engineered, synthesized, and annealed under similar conditions similar to those used for siRNA molecules. As a non-limiting example, an aiRNA sequence may be selected and generated using the methods described above to select siRNA sequences.

In yet another embodiment, the amino acid sequence of the amino acid sequence of the present invention may be of variable length (e.g., about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 base pairs, 15, 16, 17, 18, 19, or 20 base pairs) of the aiRNA duplex can be designed to have an overhang at the 3 'and 5' ends of the antisense strand to target the mRNA of interest. In certain instances, the sense strand of the aiRNA molecule is about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 nucleotides in length, more usually 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In certain other examples, the antisense strand of the aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotides in length, more typically about 15-30, 15-25, or 19-25 nucleotides in length, Is about 20-24, 21-22, or 21-23 nucleotides in length.

In some embodiments, the 5 'antisense overhang contains 1, 2, 3, 4, or more untargeted nucleotides (e.g., "AA", "UU", "dTdT", etc.). In other embodiments, the 3 'antisense overhang contains 1, 2, 3, 4, or more untargeted nucleotides (e.g., "AA", "UU", "dTdT", etc.). In particular aspects, the aiRNA molecules described herein may comprise, for example, one or more modified nucleotides within a double stranded (duplex) region and / or within an antisense overhang. By way of non-limiting example, an aiRNA sequence may comprise one or more of the modified nucleotides described above for siRNA sequences. In a preferred embodiment, the aiRNA molecule comprises a 2'OMe nucleotide, such as a 2'OMe-guanosine nucleotide, a 2'OMe-uridine nucleotide, or a mixture thereof.

In certain embodiments, the aiRNA molecule may comprise an siRNA molecule, for example, an antisense strand corresponding to the antisense strand of one siRNA molecule described herein. In certain embodiments, the aiRNA of the invention can silence ALDH gene expression. In certain embodiments, the aiRNA targeting ALDH mRNA is administered using a carrier system, such as nucleic acid-lipid particles. In a preferred embodiment, the nucleic acid-lipid particle comprises (a) at least one aiRNA molecule that targets ALDH gene expression; (b) cationic lipids (e. g., DLinDMA, DLenDMA, DLin-K-C2-DMA, and / or y-DLenDMA); And (c) non-cationic lipids (e.g., DPPC, DSPC, DSPE, and / or cholesterol). In certain cases, the nucleic acid-lipid particles may further comprise a conjugated lipid (e. G., PEG-DAA and / or POZ-DAA) that prevents aggregation of the particles.

Suitable aiRNA sequences can be identified, synthesized, and modified using any means known in the art for designing, synthesizing, and modifying siRNA sequences. Additional embodiments relating to the aiRNA molecules of the invention are described in U.S. Patent Application 12 / 343,342 (filed December 23, 2008), and U.S. Patent Application 12 / 424,367 (2009), the entirety of which is incorporated herein by reference in its entirety Filed April 15, 2005).

E. miRNA

Generally, microRNAs (miRNAs) are single-stranded RNA molecules of about 21-23 nucleotides in length that regulate gene expression. miRNAs are encoded by genes transcribed from their DNA, but miRNAs are not translated into proteins (non-coding RNAs); Instead, each primary transcript (pri-miRNA) is processed into a short stem-loop structure, termed pre-miRNA, and eventually processed into functional mature miRNA. A mature miRNA molecule is partially or completely complementary to one or more messenger RNA (mRNA) molecules, and their primary function is to down-regulate gene expression. Identification of miRNA molecules is described, for example, by Lagos-Quintana et al., Science, 294: 853-858; [Lau et al., Science, 294: 858-862]; And Lee et al., Science, 294: 862-864.

The gene encoding the miRNA is much longer than the mature miRNA molecule being treated. miRNAs are first transcribed as primary transcripts or pri-miRNAs with caps and poly-A tails and processed into short, ~ 70 nucleotide stem-loop structures known as pre-miRNAs in the cell nucleus. The treatment is performed by a protein complex known as a microprocessor complex consisting of Nuclease Drosha and double-stranded RNA binding protein Pasha in animals (Denli et al., Nature 432 : 231-235 (2004)). These pre-miRNAs are then treated with mature miRNAs in the cytoplasm by interaction with endonuclease dicers that also initiate the formation of RNA-induced silencing complexes (RISC) (Bernstein et al., Nature, 409: 363-366 (2001)). The sense strand or antisense strand of DNA can function as a template for generating miRNA.

When the dicer cleaves the pre-miRNA stem-loop, two complementary short RNA molecules are formed, but only one is integrated into the RISC complex. The strand is known as a guide strand and is selected by the argonaute protein which is a catalytically active RNase in the RISC complex on the basis of the stability of the 5 ' end (Preall et al., Curr. Biol, 16: 530-535 (2006)). The remaining strands (known as anti-guide or passenger strands) are degraded as RISC complex substrates (Gregory et al., Cell, 123: 631-640 (2005)). After integration into the active RISC complex, miRNA bases pair with their complementary mRNA molecules and induce target mRNA degradation and / or translation silencing.

The mammalian miRNA molecule is generally complementary to a site within the 3 ' UTR of the target mRNA sequence. In certain cases, the annealing of the miRNA to the target mRNA inhibits protein translation by blocking the protein translation machinery. In certain other examples, the annealing of the miRNA to the target mRNA facilitates cleavage and degradation of the target mRNA through a process similar to RNA interference (RNAi). In addition, miRNAs can target methylation of the genomic region corresponding to the targeted mRNA. In general, miRNAs function together with a complement of proteins, collectively referred to as miRNPs.

In particular aspects, the miRNA molecules described herein can be about 15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotide lengths, About 15-30, 15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or 21-23 nucleotides in length. In certain other aspects, the miRNA molecule may comprise one or more modified nucleotides. By way of non-limiting example, a miRNA sequence may comprise one or more of the modified nucleotides described above for siRNA sequences. In a preferred embodiment, the miRNA molecule comprises a 2'OMe nucleotide, such as a 2'OMe-guanosine nucleotide, a 2'OMe-uridine nucleotide, or a mixture thereof.

In some embodiments, miRNA molecules can be used to silence ALDH gene expression. In certain embodiments, miRNAs that target ALDH mRNA are administered using a carrier system, such as nucleic acid-lipid particles. In a preferred embodiment, the nucleic acid-lipid particle comprises (a) one or more miRNA molecules that target ALDH gene expression; (b) cationic lipids (e. g., DLinDMA, DLenDMA, DLin-K-C2-DMA, and / or y-DLenDMA); And (c) non-cationic lipids (e.g., DPPC, DSPC, DSPE, and / or cholesterol). In certain instances, the nucleic acid-lipid particle additionally comprises a conjugated lipid (e. G., PEG-DAA and / or POZ-DAA) that prevents aggregation of the particles.

In another embodiment, one or more agents that block the activity of an miRNA that targets ALDH mRNA is administered using the lipid particles of the invention (e.g., nucleic acid-lipid particles). Examples of blocking agents include, but are not limited to, sterically hindered oligonucleotides, locked nucleic acid oligonucleotides, and morpholino oligonucleotides. Such blocking agents may bind directly to the miRNA or to the miRNA binding site on the target RNA.

V. Compounds containing therapeutic nucleic acids carrier  system

In one aspect, the present invention provides a carrier system containing one or more therapeutic nucleic acids (e. G., Interfering RNA, e. In some embodiments, the carrier system comprises a lipid-based carrier system such as lipid particles (e.g., SNALP), a cationic lipid or a liposomal nucleic acid complex (i.e., lipoplex), liposomes, micelles, Or a mixture thereof. In another embodiment, the carrier system is a polymer-based carrier system, such as a cationic polymer-nucleic acid complex (i. E., Polyplex). In a further embodiment, the carrier system is a cyclodextrin-based carrier system, such as a cyclodextrin polymer-nucleic acid complex. In a further embodiment, the carrier system is a protein-based carrier system, such as a cationic peptide-nucleic acid complex. Preferably, the carrier system is a lipid particle, such as SNALP. Those skilled in the art will understand that the therapeutic nucleic acid of the present invention may also be delivered as a naked molecule.

A. lipid particles

In particular aspects, the invention provides lipid particles comprising at least one therapeutic nucleic acid (e.g., an interfering RNA, such as a dsRNA) and at least one cationic (amino) lipid or salt thereof. In some embodiments, the lipid particles of the present invention further comprise at least one non-cationic lipid. In another embodiment, the lipid particles further comprise at least one conjugated lipid capable of reducing or inhibiting particle aggregation.

The lipid particles of the present invention preferably comprise therapeutic nucleic acids, such as interfering RNA (e.g., siRNA), cationic lipids, non-cationic lipids, and conjugated lipids that inhibit aggregation of the particles. In some embodiments, the therapeutic nucleic acid is fully encapsulated within the lipid portion of the lipid particle such that the therapeutic nucleic acid in the lipid particle is resistant to nuclease degradation in aqueous solution. In another embodiment, the lipid particles described herein are substantially non-toxic to mammals, such as humans. The lipid particles of the present invention typically have an average diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, Or from about 70 to about 90 nm. The lipid particles of the present invention also generally have a lipid: therapeutic (e.g., lipid: nucleic acid) ratio (mass / mass ratio) of from about 1: 1 to about 100: 1, from about 1: 1 to about 50: 1 to about 25: 1, from about 3: 1 to about 20: 1, from about 5: 1 to about 15: 1, or from about 5: 1 to about 10: 1.

In a preferred embodiment, the lipid particles of the present invention comprise at least one of an interfering RNA (e.g., a dsRNA, such as an siRNA, a dicer-substrate dsRNA, a shRNA, an aRNA, and / or a miRNA), a cationic lipid (E.g., one or more cationic lipids of Formula I-III or salts thereof), non-cationic lipids (e.g., a mixture of one or more phospholipids and cholesterol) PEG-lipid conjugate), which is stable in serum. The SNALP may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more unmodified and / or modified interfering RNAs For example, siRNA). Nucleic acid-lipid particles and methods for their manufacture are described, for example, in U.S. Patent Nos. 5,753,613; 5,785,992; 5,705,385; 5,976,567; 5,981,501; 6,110,745; And 6,320,017; And PCT Publication WO 96/40964.

In the nucleic acid-lipid particle of the present invention, the nucleic acid may be completely encapsulated within the lipid portion of the particle to protect the nucleic acid from neclease decomposition. In a preferred embodiment, a SNALP comprising a nucleic acid such as an interfering RNA is fully encapsulated within the lipid portion of the particle to protect the nucleic acid from nuclease degradation. In certain cases, nucleic acids in SNALP do not substantially degrade after exposure of the particles to nuclease at 37 0 C for at least about 20, 30, 45, or 60 minutes. In certain other examples, the nucleic acid in the SNALP can be generated by incubating the particles in serum for at least about 30, 45, or 60 minutes or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, After incubation for 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In another embodiment, the nucleic acid is complexed with the lipid moiety of the particle. One of the advantages of the formulations of the present invention is that the nucleic acid-lipid particle composition is substantially non-toxic to mammals, such as humans.

The term "fully encapsulated" indicates that the nucleic acid in the nucleic acid-lipid particle is not significantly degraded after exposure to serum or nuclease assays to significantly degrade free DNA or RNA. In a fully encapsulated system, normally less than about 25% of the nucleic acids in the particle are degraded in a treatment that degrades 100% of the free nucleic acid, more preferably less than about 10%, most preferably less than about 5 % Of nucleic acids are degraded. "Fully encapsulated" also indicates that the nucleic acid-lipid particles are stable in serum, i.e., the particles are not rapidly degraded into their component parts in vivo administration.

In terms of nucleic acids, complete encapsulation can be determined by performing a membrane-impermeable fluorescent dye exclusion assay using a dye that exhibits enhanced fluorescence when associated with nucleic acids. A specific dye, e.g., post-drawn (OliGreen) ^® and ribonucleic green (RiboGreen) ^® (Invitrogen Corporation (Invitrogen Corp.); Carlsbad, Calif.) Plasmid DNA, single-stranded deoxyribonucleic nucleotides, and / or single- Or for quantitative determination of double-stranded ribonucleotides. Encapsulation is determined by adding a dye to the liposomal formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon the addition of a small amount of non-ionic detergent. Detergent-mediated destruction of the liposomal bilayer releases the encapsulated nucleic acid, whereby the nucleic acid can interact with the membrane-impermeable dye. Is a nucleic acid encapsulated can be calculated by the following _{equation:. E = (I o -} I) / I o ( here, I / I _o is being fluorescence intensity before and after the addition of the detergent) (lit. [Wheeler et al, Gene Ther. , 6: 271-281 (1999)).

In another embodiment, the present invention provides a nucleic acid-lipid particle (e.g., SNALP) composition comprising a plurality of nucleic acid-lipid particles.

In some cases, the SNALP composition comprises about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100% About 90% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, about 70% About 95%, about 95%, about 90% to about 95%, about 30% to about 90%, about 40% to about 90%, about 50% , About 60% to about 90%, about 70% to about 90%, about 80% to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55% (Or any ratio thereof) of at least 70%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or ranges within) a nucleic acid that is completely encapsulated within the lipid portion of the particle such that the particle has an encapsulated nucleic acid therein.

In another example, the SNALP composition comprises about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100% About 90% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, about 70% About 95%, about 95%, about 90% to about 95%, about 30% to about 90%, about 40% to about 90%, about 50% , About 60% to about 90%, about 70% to about 90%, about 80% to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55% (Or any ratio thereof) of at least 70%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or ranges within) a nucleic acid that is completely encapsulated within the lipid portion of the particle so that the input nucleic acid is encapsulated within the particle.

Depending on the intended use of the lipid particles of the present invention, the proportions of the components may be different and the delivery efficiency of the particular agent may be determined, for example, using endosomal release parameter (ERP) assays.

One. Cationic  Lipid

Any of a variety of cationic lipids or salts thereof may be used in the lipid particles (e.g., SNALP) of the present invention, alone or in combination with one or more other cationic lipid species or non-cationic lipid species. The cationic lipid comprises its (R) and / or (S) enantiomer.

In one aspect, the cationic lipids of formula (I) or salts thereof having the following structure are useful in the present invention:

(I)

Wherein R ¹ and R ² are the same or different and are independently hydrogen (H) or optionally substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, or C ₂ -C ₆ alkynyl, or R ¹ and R ² are connected together to form an optionally substituted heterocycle ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and mixtures thereof Can be;

R ³ is absent or is C ₁ -C ₆ alkyl to provide hydrogen (H) or a quaternary amine;

R ⁴ and R ⁵ are the same or different and are independently optionally substituted C ₁₀ -C ₂₄ alkyl, C ₁₀ -C ₂₄ alkenyl, C ₁₀ -C ₂₄ alkynyl, or C ₁₀ -C ₂₄ acyl, wherein R ⁴ and R < ⁵ > comprises at least two unsaturated moieties;

n is 0, 1, 2, 3, or 4;

In some embodiments, R ¹ and R ² are independently optionally substituted C ₁ -C ₄ alkyl, C ₂ -C ₄ alkenyl, or C ₂ -C ₄ alkynyl. In one preferred embodiment, R ¹ and R ² are both methyl groups. In another preferred embodiment, n is 1 or 2. In another embodiment, R ³ is absent when the pH exceeds the pKa of the cationic lipid and R ³ is hydrogen when the pH is below the pKa of the cationic lipid so that the aminohead group is protonated. In another embodiment, R ³ is optionally substituted C ₁ -C ₄ alkyl to provide a quaternary amine. In a further embodiment, R ⁴ and R ⁵ are independently optionally substituted C ₁₂ -C ₂₀ or C ₁₄ -C ₂₂ alkyl, C ₁₂ -C ₂₀ or C ₁₄ -C ₂₂ alkenyl, C ₁₂ -C ₂₀ or C ₁₄ -C ₂₂ alkynyl, or C ₁₂ -C ₂₀ or C ₁₄ -C ₂₂ acyl, wherein at least one of R ⁴ and R ⁵ comprises at least two unsaturated moieties.

In certain embodiments, R ⁴ and R ⁵ are selected from the group consisting of dodecadienyl moiety, tetradecadienyl moiety, hexadecadienyl moiety, octadecadienyl moiety, eicosadienyl moiety, dodecatrienyl Octadecanetrienyl moiety, octadecanetrienyl moiety, arachidonyl moiety, and docosahexaenoyl moiety, tetradecanyl moiety, hexadecanetrienyl moiety, octadecanetrienyl moiety, And acyl derivatives thereof (for example, linoleoyl, linolenolyl,? -Linolenyl and the like). In some instances, one of R ⁴ and R ⁵ includes a branched alkyl group (e.g., a pyranyl moiety) or an acyl derivative thereof (e.g., a pyranyl moiety). In certain cases, the octadecadienyl moiety is a linoleyl moiety. In certain other examples, the octadecatrienyl moiety is a linolenyl moiety or a? -Linolenyl moiety. In certain embodiments, R ⁴ and R ⁵ are both a linoleyl moiety, a linolenyl moiety, or a? -Linolenyl moiety. In certain embodiments, the cationic lipid of Formula I is selected from the group consisting of 1,2-dirinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N-dimethylaminopropane ), 1,2-dirinoleyloxy- (N, N-dimethyl) -butyl-4-amine (C2-DLinDMA), 1,2- 4-amine (C2-DLinDAP), or a mixture thereof.

In some embodiments, the cationic lipids of formula I form salts (preferably crystalline salts) with one or more cations. In one particular embodiment, the cationic lipid of formula I is its oxalate (e.g., hemioxalate) salt, which is preferably a crystalline salt.

The synthesis of cationic lipids such as DLinDMA and DLenDMA, and additional cationic lipids is described in U.S. Patent Publication 20060083780, the full text of which is incorporated herein by reference in its entirety. The synthesis of cationic lipids such as C2-DLinDMA and C2-DLinDAP, and additional cationic lipids is described in international patent application WO2011 / 000106, the full text of which is incorporated herein by reference in its entirety.

In another aspect, cationic lipids of formula (II) (or salts thereof) having the following structure are useful in the present invention:

≪

Wherein R ¹ and R ² are the same or different and are independently optionally substituted C ₁₂ -C ₂₄ alkyl, C ₁₂ -C ₂₄ alkenyl, C ₁₂ -C ₂₄ alkynyl, or C ₁₂ -C ₂₄ acyl ; R ³ and R ⁴ are the same or different and are independently optionally substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, or C ₂ -C ₆ alkynyl, or R ³ and R ⁴ are linked together 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from nitrogen and oxygen; R ⁵ is absent, or hydrogen (H) or a quaternary C ₁ -C ₆ alkyl to give an amine; m, n, and p are the same or different and independently 0, 1, or 2, with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; Y and Z are the same or different and independently O, S, or NH. In a preferred embodiment, q is 2.

In some embodiments, the cationic lipid of Formula II is 2,2-dirinoleyl-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (DLin-K- C3-DMA; "C3K"), 2,2-dirinoleyl-4- (3-dimethylaminopropyl) (DLin-K-C4-DMA; "C4K"), 2,2-dirinoleyl-5-dimethylaminomethyl- [1,3] -dioxane (DLin-K6-DMA), 2,2-dirinoleyl-4-N-methylpepiazino- [ Dimethyl-aminomethyl- [1,3] -dioxolane (DLin-K-DMA), 2,2-diolureo-4-dimethylaminomethyl- [1,3] (D-K-DMA), 2,2-distearoyl-4-dimethylaminomethyl- [1,3] -dioxolane (DS- -Morpholino- [1,3] -dioxolane (DLin-K-MA), 2,2-dirinoleyl-4-trimethylamino- [1,3] -dioxolane chloride (DLin-K-TMA. Cl), 2,2-dirinoleyl-4,5-bis (dimethylaminomethyl) - [1,3] Is ^{(DLin-K 2 -DMA),} 2,2- di-linoleyl-4-methylpiperidin-Jean Fernand [1, 3] - dioxolane (DLin-KN- methylpiperidin-percha binary), or mixtures thereof to be. In a preferred embodiment, the cationic lipid of formula II is DLin-K-C2-DMA.

In some embodiments, the cationic lipids of formula (II) form salts (preferably crystalline salts) with one or more anions. In one particular embodiment, the cationic lipid of formula (II) is its oxalate (e.g., hemioxalate) salt, which is preferably a crystalline salt.

The synthesis of cationic lipids such as DLin-K-DMA, and additional cationic lipids is described in PCT Publication No. WO 09/086558, the full text of which is incorporated herein by reference in its entirety. D-K-DMA, DL-K-DMA, DLin-K-DMA, DLin-K-MPZ, The synthesis of DL-K-MA, DLin-K-TMA.Cl, DLin-K ² -DMA, and D-Lin-KN-methyl piperazine, and additional cationic lipids, PCT Application No. PCT / US2009 / 060251, entitled " Improved Amino Lipids and Methods for the Delivery of Nucleic Acids, " filed October 9, 2009, which is incorporated herein by reference in its entirety for purposes.

In a further aspect, a cationic lipid of formula (III), or salt thereof, having the following structure is useful in the present invention:

(III)

Wherein R ¹ and R ² are the same or different and are independently optionally substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, or C ₂ -C ₆ alkynyl, or R ¹ and R ² are Taken together may form an optionally substituted heterocycle ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and mixtures thereof; R ³ is absent or is C ₁ -C ₆ alkyl to provide hydrogen (H) or a quaternary amine; R ⁴ and R ⁵ are not present or present and when present on the same or different and independently represent an optionally substituted C ₁ -C ₁₀ alkyl or C ₂ -C ₁₀ alkenyl; n is 0, 1, 2, 3, or 4;

In some embodiments, R ¹ and R ² are independently optionally substituted C ₁ -C ₄ alkyl, C ₂ -C ₄ alkenyl, or C ₂ -C ₄ alkynyl. In a preferred embodiment, R ¹ and R ² are both methyl groups. In another preferred embodiment, R ⁴ and R ⁵ are both butyl groups. In another preferred embodiment, n is 1. In another embodiment, R ³ is absent when the pH exceeds the pKa of the cationic lipid and R ³ is hydrogen when the pH is below the pKa of the cationic lipid so that the aminohead group is protonated. In another embodiment, R ³ is optionally substituted C ₁ -C ₄ alkyl to provide a quaternary amine. In a further embodiment, R ⁴ and R ⁵ are independently optionally substituted C ₂ -C ₆ or C ₂ -C ₄ alkyl or C ₂ -C ₆ or C ₂ -C ₄ alkenyl.

In another embodiment, the cationic lipid of formula (III) comprises an ester linkage between the aminohead group and one or both alkyl chains. In some embodiments, the cationic lipid of formula (III) forms a salt (preferably a crystalline salt) with at least one anion. In one particular embodiment, the cationic lipid of formula (III) is its oxalate (e.g., hemioxalate) salt, which is preferably a crystalline salt.

Each alkyl chain in Formula III contains cis double bonds (i.e., cis, cis, cis-Δ ⁶ , Δ ⁹ , Δ ¹² ) at positions 6, 9, and 12, while in other embodiments, one or both One, two, or three of these double bonds in the alkyl chain of the alkyl moiety may be present in the trans-isomer form.

In a particularly preferred embodiment, the cationic lipid of formula (III) has the structure:

The synthesis of cationic lipids, such as y-DLenDMA, and additional cationic lipids is described in U. S. Patent Application Serial No. 61 / 222,462, entitled "Improved Cationic Lipids and Methods for the Delivery of Nucleic Acids ", filed on July 1, 2009).

In certain embodiments, cationic lipids having the following structure are useful in the present invention:

The synthesis of cationic lipids such as DLin-M-C3-DMA ("MC3"), and additional cationic lipids (eg, specific analogs of MC3) is described in US Pat. U.S. Provisional Patent Application 61 / 185,800 entitled " Novel Lipids and Compositions for the Delivery of Therapeutics ", filed on June 10, 2009, and U.S. Provisional Patent Application 61 / 287,995 of Nucleic Acids ", filed December 18, 2009).

Examples of other cationic lipids or salts thereof that may be included in the lipid particles of the present invention include cationic lipids such as those described in WO2011 / 000106, which is incorporated herein by reference in its entirety for all purposes, and cationic lipids such as N (DODAC), 1,2-dioleyloxy-N, N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N, N-dimethyl Aminopropane (DSDMA), N- (1- (2,3-diolyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTMA), N, N-distearyl- N, N, N-trimethylammonium chloride (DOTAP), 3- (N- (N ', N'-dimethylamino N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-dimethylformamide (DMRIE) 2,3-diolyloxy-N- [2 (spermine-carboxamido) ethyl] -N, N-di (DOSPA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2- (cholest-5-ene-3-beta-oxybutane (Cholest-5-ene-3-beta-oxy) -3 ', 5'-bipyridyloxy) (CpLinDMA), N, N-dimethyl-3, 4-dioleyloxybenzylamine (DMOBA), 1,2-N, N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N, N'-dirinoleylcarbamyl- (DLin-C-DAP), 1,2-dirinoleoyloxy-3- (dimethylamino) acetoxypropane (DLin-DAC), 1,2-dirinoleylcarbamoyloxy- (DLin-MA), 1,2-dirinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dirinoleylthio-3- Dimethylaminopropane (DLin-S-DMA), 1- (DLin-2-DMAP), 1,2-dirinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2 (DLin-TAP.Cl), 1,2-dirinoleyloxy-3- (N-methylpiperazino) propane (DLin-MPZ), 3- N, N-dirinoleylamino) -1,2-propanediol (DLinAP), 3- (N, N-dioleylamino) -1,2-propanedio (DOAP) (2-N, N-dimethylamino) ethoxypropane (DLin-EG-DMA), 1,2-dioleylcarbamoyloxy- Dimyristoleoleyl-3-dimethylaminopropane (DMDAP), 1,2-diolureo-3-trimethylaminopropane chloride (DOTAP.Cl), dirinoleylmethyl-3-dimethylaminopropionate -M-C2-DMA; Also known as DLin-M-K-DMA or DLin-M-DMA), and mixtures thereof. Additional cationic lipids or salts thereof that may be included in the lipid particles of the present invention are described in U.S. Patent Publication No. 20090023673, the entirety of which is incorporated herein by reference in its entirety.

The synthesis of cationic lipids such as CLinDMA, and additional cationic lipids is described in U.S. Patent Publication No. 20060240554, the full text of which is incorporated herein by reference in its entirety. DLin-C-DAP, DLinDAC, DLinMA, DLinDAP, DLin-S-DMA, DLin-2-DMAP, DLinTMA.Cl, DLinTAP.Cl, DLinMPZ, DLinAP, DOAP, And the synthesis of additional cationic lipids are described in PCT Publication No. WO 09/086558, the full text of which is incorporated herein by reference for all purposes. The synthesis of cationic lipids such as DO-C-DAP, DMDAP, DOTAP.Cl, DLin-M-C2-DMA and additional cationic lipids is described in PCT application PCT / US2009 / 060251, entitled "Improved Amino Lipids and Methods for the Delivery of Nucleic Acids" filed on October 9, 2009. The synthesis of many different cationic lipids and related analogs is described in U. S. Patent Nos. 5,208, 036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; And 5,785,992; And PCT publication WO 96/10390. In addition, the many commercially available cationic agent, such as lipoic pectin (LIPOFECTIN) ^® of lipids (including DOTMA and DOPE possible available from Invitrogen); Lipofectamine (LIPOFECTAMINE) ^® (comprising DOSPA and DOPE possible available from Invitrogen); The (available comprising DOGS from Promega Corp., (Promega Corp.)), and transfected Tam (TRANSFECTAM) ^® it can be used.

In some embodiments, the cationic lipid comprises from about 50 mole% to about 90 mole%, from about 50 mole% to about 85 mole%, from about 50 mole% to about 80 mole%, from about 50 mole% To about 75 mole%, about 50 mole% to about 70 mole%, about 50 mole% to about 65 mole%, about 50 mole% to about 60 mole%, about 55 mole% to about 65 mole%, or about 55 mole% % To about 70 mole% (or any range or range thereof). In certain embodiments, the cationic lipids comprise about 50 mole%, 51 mole%, 52 mole%, 53 mole%, 54 mole%, 55 mole%, 56 mole%, 57 mole%, 58 mole%, 58 mole% 59 mole%, 60 mole%, 61 mole%, 62 mole%, 63 mole%, 64 mole%, or 65 mole% (or any proportion thereof).

In another embodiment, the cationic lipid comprises from about 2 mol% to about 60 mol%, from about 5 mol% to about 50 mol%, from about 10 mol% to about 50 mol%, to about 20 mol% To about 50 mole%, about 20 mole% to about 40 mole%, about 30 mole% to about 40 mole%, or about 40 mole% (or any range or range thereof).

Additional proportions and ranges of cationic lipids suitable for use in the lipid particles of the present invention are described in PCT Publication No. WO 09/127060, U.S. Pat. U.S. Patent Application Publication No. US 2011/0071208, PCT Publication No. WO2011 / 000106, and U.S. Pat. Patent application publication US 2011/0076335.

It should be understood that the ratio of cationic lipids present in the lipid particles of the present invention is a target amount and that the actual amount of cationic lipid present in the formulation may differ, for example, by +/- 5 mol%. For example, in a 1:57 lipid particle (e.g., SNALP) formulation, the target amount of cationic lipid is 57.1 mole%, but the actual amount of cationic lipid is within ± 5 mole%, +/- 4 mole% It should be appreciated that the formulation may be 3 mol%, ± 2 mol%, ± 1 mol%, ± 0.75 mol%, ± 0.5 mol%, ± 0.25 mol%, or ± 0.1 mol% (Up to 100 mol% of the total lipid present in the particles).

2. Non- Cationic  Lipid

Non-cationic lipids used in the lipid particles (e.g., SNALP) of the present invention may be any of a variety of neutral, uncharged, zwitterionic, or anionic lipids capable of producing a stable complex.

Non-limiting examples of non-cationic lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM) (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioloylphosphatidylglycerol (dicyclohexylphosphatidylglycerol, dicyclohexylphosphatidylglycerol, dicyclohexylphosphatidylglycerol, DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioloylphosphatidylethanolamine (DOPE), palmitoyl oleoyl-phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), palmitoyl oleyl-phosphatidyl Glycerol (POPG), dioloylphosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl- Phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, Phosphatidylethanolamine (DEPE), stearoylooleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dirinoleoylphosphatidylcholine, and mixtures thereof. Other diacyl phosphatidylcholine and diacyl phosphatidylethanolamine phospholipids may also be used. The acyl group in these lipids is preferably an acyl group derived from a fatty acid having a C ₁₀ -C ₂₄ carbon chain, for example, lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

Further examples of non-cationic lipids include sterols, such as cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 5β-coprostanol, cholesteryl- (2'-hydroxy) -ethylether, cholesteryl- (4'- ) -Butyl ether, and 6-ketolestestanol; Non-polar analogs such as 5α-cholestane, cholestenone, 5α-cholestarone, 5β-cholestenone, and cholesteryl decanoate; And mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analog such as cholesteryl- (4 ' -hydroxy) -butyl ether. The synthesis of cholesteryl- (2 ' -hydroxy) -ethyl ether is described in PCT Publication WO 09/127060, the full text of which is incorporated herein by reference for all purposes.

In some embodiments, the non-cationic lipid present in the lipid particles (e. G., SNALP) comprises or consists of a mixture of one or more phospholipids and cholesterol or derivatives thereof. In another embodiment, the non-cationic lipid present in the lipid particles (e. G., SNALP) comprises or consists of one or more phospholipids, e. G., Lipid particle formulation without cholesterol. In another embodiment, the non-cationic lipid present in lipid particles (e. G., SNALP) comprises or consists of cholesterol or derivatives thereof, for example, a lipid particle formulation free of phospholipids.

Other examples of suitable non-cationic lipids for use herein include non-phosphorus containing lipids such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, iso Propyl myristate, amphoteric acrylic polymer, triethanolamine-lauryl sulfate, alkyl-arylsulfate polyethyloxylated fatty acid amide, dioctadecyldimethylammonium bromide, ceramide, sphingomyelin and the like.

In some embodiments, the non-cationic lipid comprises about 10 mole% to about 60 mole%, about 20 mole% to about 55 mole%, about 20 mole% to about 45 mole%, about 20 mole% From about 30 mol% to about 45 mol%, from about 30 mol% to about 45 mol%, from about 25 mol% to about 50 mol%, from about 25 mol% About 35 mol% to about 45 mol%, about 37 mol% to about 42 mol%, or about 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol% 40 mole%, 41 mole%, 42 mole%, 43 mole%, 44 mole%, or 45 mole% (or any range or range thereof).

In embodiments where the lipid particles contain a mixture of phospholipids and cholesterol or cholesterol derivatives, the mixture may comprise up to about 40 mole%, 45 mole%, 50 mole%, 55 mole%, or 60 mole% of the total lipid present in the particles can do.

In some embodiments, the phospholipid component in the mixture comprises from about 2 mol% to about 20 mol%, from about 2 mol% to about 15 mol%, from about 2 mol% to about 12 mol%, from about 4 mol % To about 15 mole%, or from about 4 mole% to about 10 mole% (or any range or range thereof). In certain preferred embodiments, the phospholipid component in the mixture comprises from about 5 mol% to about 10 mol%, from about 5 mol% to about 9 mol%, from about 5 mol% to about 8 mol%, from about 5 mol% To about 9 mol%, about 6 mol% to about 8 mol%, or about 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, or 10 mol% Or a range within it). By way of non-limiting example, a 1:57 lipid particle formulation comprising a mixture of phospholipids and cholesterol may be prepared, for example, with about 34 mole% (or any proportion thereof) of the total lipid present in the particles, with a cholesterol or cholesterol derivative The phospholipid, such as DPPC or DSPC, can be included in the mixture at about 7 mole% (or any proportion thereof). As another non-limiting example, a 7:54 lipid particle formulation comprising a mixture of phospholipids and cholesterol can be prepared, for example, from about 32 mole% (or any proportion thereof) of total lipid present in the particles, cholesterol or cholesterol derivative (E.g., DPPC or DSPC) in the mixture with about 7 mol% (or any proportion thereof) of a phospholipid such as DPPC or DSPC.

In another embodiment, the cholesterol component in the mixture comprises from about 25 mol% to about 45 mol%, from about 25 mol% to about 40 mol%, from about 30 mol% to about 45 mol%, from about 30 mol% To about 40 mole%, from about 27 mole% to about 37 mole%, from about 25 mole% to about 30 mole%, or from about 35 mole% to about 40 mole% (or any range or range thereof) can do. In certain preferred embodiments, the cholesterol component in the mixture comprises from about 25 mol% to about 35 mol%, from about 27 mol% to about 35 mol%, from about 29 mol% to about 35 mol%, from about 30 mol% From about 30 mol% to about 34 mol%, from about 31 mol% to about 33 mol%, or from about 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol% Or 35 mole% (or any range or range thereof). Generally, a 1:57 lipid particle formulation comprising a mixture of phospholipids and cholesterol can be prepared, for example, in a mixture with phospholipids, such as DPPC or DSPC, at about 7 mole percent (or any proportion thereof) Cholesterol or cholesterol derivative at about 34 mole% (or any proportion thereof). Generally, a 7:54 lipid particle formulation comprising a mixture of phospholipids and cholesterol can be prepared, for example, in a mixture with phospholipids, such as DPPC or DSPC, at about 7 mole% (or any proportion thereof) Cholesterol or cholesterol derivative at about 32 mol% (or any proportion thereof).

In embodiments where the lipid particles are free of phospholipids, the cholesterol or derivative thereof may be present in the particles in an amount of about 25 mole%, 30 mole%, 35 mole%, 40 mole%, 45 mole%, 50 mole%, 55 mole% , Or 60 mol%.

In some embodiments, the cholesterol or derivative thereof in the phospholipid-free lipid particle formulation comprises from about 25 mol% to about 45 mol%, from about 25 mol% to about 40 mol%, from about 30 mol% to about 45 mol% 45 mol%, about 30 mol% to about 40 mol%, about 31 mol% to about 39 mol%, about 32 mol% to about 38 mol%, about 33 mol% to about 37 mol%, about 35 mol% 45 mol%, about 35 mol% to about 35 mol%, or about 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol% 36 mol%, 37 mol%, 38 mol%, 39 mol%, or 40 mol% (or any range or range thereof). As a non-limiting example, a 1:62 lipid particle formulation can contain cholesterol at about 37 mole% (or any proportion thereof) of the total lipid present in the particle. As another non-limiting example, a 7:58 lipid particle formulation can contain cholesterol at about 35 mole% (or any proportion thereof) of the total lipid present in the particles.

In another embodiment, the non-cationic lipid comprises from about 5 mol% to about 90 mol%, from about 10 mol% to about 85 mol%, from about 20 mol% to about 80 mol%, from about 10 mol% (E.g., phospholipids alone), or about 60 mole% (e.g., phospholipids and cholesterol or derivatives thereof) (or ranges or ranges thereof).

Additional proportions and ranges of non-cationic lipids suitable for use in the lipid particles of the present invention are described in PCT Publication No. WO 09/127060, U.S. Pat. U.S. Patent Application Publication No. US 2011/0071208, PCT Publication No. WO2011 / 000106, and U.S. Pat. Patent application publication US 2011/0076335.

It is to be understood that the ratio of non-cationic lipids present in the lipid particles of the present invention is a target amount and that the actual amount of non-cationic lipids present in the formulation may differ, for example, by +/- 5 mole%. For example, in a 1:57 lipid particle (e.g., SNALP) preparation, the target amount of phospholipid is 7.1 mol% and the target amount of cholesterol is 34.3 mol%, but the actual amount of phospholipid is ± 2 mol% And the actual amount of cholesterol is ± 3 mol%, ± 2 mol%, ± 1 mol% of the target amount, and the actual amount of cholesterol is ± 1 mol%, ± 0.75 mol%, ± 0.5 mol%, ± 0.25 mol% %, ± 0.75 mol%, ± 0.5 mol%, ± 0.25 mol%, or ± 0.1 mol% of the total lipid content Is added. Similarly, in a 7:54 lipid particle (e.g., SNALP) preparation, the target amount of phospholipid is 6.75 mol% and the target amount of cholesterol is 32.43 mol%, but the actual amount of phospholipid is within ± 2 mol% The actual amount of cholesterol may be within ± 3 mol%, ± 2 mol%, ± 2 mol% of the target amount, 1 mol%, ± 0.75 mol%, ± 0.5 mol%, ± 0.25 mol%, or ± 0.1 mol%, where the remainder of the formulation contains other lipid components (up to 100 mol% ).

3. Lipid junction

In addition to cationic and non-cationic lipids, the lipid particles of the present invention (e. G., SNALP) may further comprise a lipid conjugate. The conjugated lipids are useful in that they prevent aggregation of the particles. Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, POZ-lipid conjugates, ATTA-lipid conjugates, cationic-polymer-lipid conjugates (CPL), and mixtures thereof. In certain embodiments, the particle comprises a PEG-lipid conjugate or an ATTA-lipid conjugate with CPL.

In a preferred embodiment, the lipid conjugate is a PEG-lipid. Examples of PEG-lipids include, for example, PEG (PEG-DAA) conjugated to a dialkyloxypropyl as described in PCT Publication No. WO 05/026372, such as those conjugated to diacylglycerol described in U.S. Patent Nos. 20030077829 and 2005008689 PEG (PEG-PE) conjugated to PEG (PEG-DAG), a phospholipid such as phosphatidylethanolamine, PEG conjugated to ceramides as described for example in US 5,885,613, PEG conjugated to cholesterol or derivatives thereof, Mixtures thereof, and the like. The disclosure of which is incorporated herein by reference in its entirety for all purposes.

Additional PEG-lipids suitable for use in the present invention include, but are not limited to, mPEG2000-1,2-di-O-alkyl-sn3-carbamoylglyceride (PEG-C-DOMG). The synthesis of PEG-C-DOMG is described in PCT Publication No. WO 09/086558, the full text of which is incorporated herein by reference for all purposes. Still other suitable PEG-lipid conjugates include, but are not limited to, 1- [8 '- (1,2-dimyristoyl-3-propanoxy) -carboxamide-3', 6'-dioxaoctanyl ] Carbamoyl-omega-methyl-poly (ethylene glycol) (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in U.S. Patent No. 7,404,969, the full text of which is incorporated herein by reference for all purposes.

PEG is a water soluble linear polymer of ethylene PEG repeat units having two terminal hydroxyl groups. PEGs are classified by their molecular weight; For example, PEG 2000 has an average molecular weight of about 2,000 daltons and PEG 5000 has an average molecular weight of about 5,000 daltons. PEG is commercially available from Sigma Chemical Co. and other companies and includes, but is not limited to, monomethoxy polyethylene glycol (MePEG-OH), monomethoxy polyethylene glycol-succinate (MePEG-S), mono-methoxy-polyethylene glycol-succinimidyl succinate (MePEG-S-NHS), mono-methoxy-polyethylene glycol-amine (MePEG-NH _2), mono-methoxy-polyethylene glycol-TRE salicylate (MePEG (For example, HO-PEG-S, HO-PEG-S-TRES), monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM), and a compound containing a terminal hydroxyl group in place of a terminal methoxy group PEG-S-NHS, HO- PEG-NH 2 , etc.). Other PEGs such as those described in U.S. Patent Nos. 6,774,180 and 7,053,150 (e.g., mPEG (20 KDa) amine) are also useful for preparing the PEG-lipid conjugates of the present invention. The disclosures of these patent documents are incorporated herein by reference in their entirety for all purposes. In addition, monomethoxypolyethylene glycol-acetic acid (MePEG-CH ₂ COOH) is particularly useful for preparing PEG-lipid conjugates including, for example, PEG-DAA conjugates.

The average molecular weight of the PEG moieties of the PEG-lipid conjugates described herein can range from about 550 daltons to about 10,000 daltons. In certain instances, the average molecular weight of the PEG moieties can range from about 750 Daltons to about 5,000 Daltons (e.g., from about 1,000 Daltons to about 5,000 Daltons, from about 1,500 Daltons to about 3,000 Daltons, from about 750 Daltons to about 3,000 Daltons, To about 2,000 daltons). In a preferred embodiment, the average molecular weight of the PEG moiety is about 2,000 daltons or about 750 daltons.

In certain instances, the PEG may be optionally substituted with an alkyl, alkoxy, acyl, or aryl group. PEG may be conjugated directly to the lipid or may be linked to the lipid through a linker moiety. Any linker moiety suitable for coupling PEG to lipids can be used, for example, an ester-free linker moiety and an ester-containing linker moiety. In a preferred embodiment, the linker moiety is an ester-free linker moiety. As used herein, the term "ester-free linker moiety" refers to a linker moiety that does not contain a carboxyl ester linkage (-OC (O) -). Suitable ester-free linker moieties include amido (-C (O) NH-), amino (-NR-), carbonyl (-C (O) -), carbamate (-NHC (O) , urea (-NHC (O) NH-), disulfide (-SS-), ether (-O-), succinyl _{(- (O) CCH 2 CH} 2 C (O) -), amido succinic dill (- NHC (O) CH ₂ CH ₂ C (O) NH-), ethers, disulfides, and combinations thereof (such as linkers that contain both carbamate linker moieties and amido linker moieties) It does not. In a preferred embodiment, a carbamate linker is used to link the PEG to the lipid.

In another embodiment, an ester containing linker moiety is used to link the PEG to the lipid. Suitable ester containing linker moieties include, for example, carbonates (-OC (O) O-), succinoyl, phosphate esters (-O- (O) POH-O-), sulfonate esters, It contains water.

Phosphatidylethanolamine having various acyl chain groups of different chain length and degree of saturation can be conjugated to PEG to form a lipid conjugate. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. Phosphatidyl-ethanolamine containing saturated or unsaturated fatty acids having a carbon chain length in the range of C ₁₀ to C ₂₀ is preferred. Phosphatidylethanolamines having a mixture of mono- or di-unsaturated fatty acids and saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include dimeristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioloylphosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE) But are not limited to these.

The term "ATTA" or "polyamide" includes, without limitation, the compounds described in U.S. Patent Nos. 6,320,017 and 6,586,559, the disclosures of which are hereby incorporated by reference in their entirety for all purposes. These compounds include compounds having the formula:

(IV)

Wherein R is a member selected from the group consisting of hydrogen, alkyl and acyl; R ¹ is a member selected from the group consisting of hydrogen and alkyl; Or optionally, R and R < ¹ > and the nitrogen to which they are attached form an azido moiety; R ² is a member of the group selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl and amino acid; R ³ is a member selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, mercapto, hydrazino, amino and NR ⁴ R ⁵ wherein R ⁴ and R ⁵ are independently hydrogen or alkyl; n is from 4 to 80; m is 2 to 6; p is 1 to 4; q is 0 or 1; It will be apparent to those skilled in the art that other polyamides can be used in the compounds of the present invention.

The term "diacylglycerol" or "DAG" has two fatty acyl chains R ¹ and R ^{2 both} independently of which have 2 to 30 carbons bound to the 1- and 2-positions of the glycerol by an ester linkage ≪ / RTI > Acyl groups may be saturated or may have different degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C ₁₂ ), myristoyl (C ₁₄ ), palmitoyl (C ₁₆ ), stearoyl (C ₁₈ ), and icosyl (C ₂₀ ). In a preferred embodiment, R ¹ and R ² are the same, ie, R ¹ and R ² are both myristoyl (ie, dimyristoyl), or R ¹ and R ² are both stearoyl , Distearoyl), and the like. Diacylglycerol includes the following general formula:

(V)

The term "dialkyloxypropyl" or "DAA" includes compounds having two alkyl chains R ¹ and R ² both having 2 to 30 carbons independently. The alkyl group may be saturated or may have a different degree of unsaturation. The dialkyloxypropyl has the general formula:

≪ Formula (VI)

In a preferred embodiment, the PEG-lipid is a PEG-DAA conjugate having the formula:

(VII)

Wherein R ¹ and R ² are independently selected and are long chain alkyl groups having from about 10 to about 22 carbon atoms; PEG is polyethylene glycol; L is an ester-free linker moiety or an ester-containing linker moiety as described above. The long chain alkyl group may be saturated or unsaturated. Suitable alkyl groups are decyl (C _10), lauryl (C _12), myristyl (C _14), palmityl (C _16), stearyl (C _18), and Ico not seal comprises a (C ₂₀₎ and limited to, Do not. In a preferred embodiment, R ¹ and R ² are the same, ie, R ¹ and R ² are both myristyl (ie, dimyristyl), or R ¹ and R ² are both stearyl Reel).

In formula (VII), the average molecular weight of the PEG is from about 550 daltons to about 10,000 daltons. In certain instances, the average molecular weight of the PEG may range from about 750 Daltons to about 5,000 Daltons (e.g., from about 1,000 Daltons to about 5,000 Daltons, from about 1,500 Daltons to about 3,000 Daltons, from about 750 Daltons to about 3,000 Daltons, from about 750 Daltons to about 2,000 daltons, etc.). In a preferred embodiment, the average molecular weight of the PEG is about 2,000 daltons or about 750 daltons. PEG may be optionally substituted with alkyl, alkoxy, acyl, or aryl groups. In certain embodiments, the terminal hydroxyl group is substituted with a methoxy or methyl group.

In a preferred embodiment, "L" is an ester-free linker moiety. Suitable non-ester containing linkers include but are not limited to amido linker moiety, amino linker moiety, carbonyl linker moiety, carbamate linker moiety, urea linker moiety, ether linker moiety, disulfide linker moiety, Linker moieties, and combinations thereof. In a preferred embodiment, the ester-free linker moiety is a carbamate linker moiety (i. E., A PEG-C-DAA conjugate). In another preferred embodiment, the ester-free linker moiety is an amido linker moiety (i. E., A PEG-DAA conjugate). In another preferred embodiment, the ester-free linker moiety is a succinimidyl linker moiety (i. E., A PEG-S-DAA conjugate).

In certain embodiments, the PEG-lipid conjugate is selected from:

PEG-DAA conjugates are synthesized using standard techniques and reagents known to those skilled in the art. It will be appreciated that the PEG-DAA conjugate will contain various amides, amines, ethers, thio, carbamates, and urea linkages. Those skilled in the art will appreciate that methods and reagents for forming such bonds are known and readily available. For example, March, ADVANCED ORGANIC CHEMISTRY (Wiley 1992); [Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989)]; And Furniss, VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th ed. (Longman 1989). It will also be appreciated that any functional groups present may require protection and deprotection at different points in the synthesis of PEG-DAA conjugates. Those skilled in the art will appreciate that the techniques are known. See, for example, Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley 1991).

Preferably, PEG-DAA conjugate is PEG- didecyl oxy profile (C ₁₀₎ conjugate, PEG- dilauryl oxy profile (C ₁₂₎ conjugate, PEG- dimyristyl oxy profile (C ₁₄₎ conjugate, palmityl di-PEG- (C ₁₆ ) conjugate, or a PEG-distearyloxypropyl (C ₁₈ ) conjugate. In these embodiments, the average molecular weight of the PEG is preferably about 750 or about 2,000 Daltons. &Quot; DMA " refers to the average molecular weight of PEG, "C" refers to carbamate linker moiety, "DMA" refers to carbamate linker moiety, Represents dimyristyloxypropyl. In another particularly preferred embodiment, the PEG-lipid conjugate comprises PEG750-C-DMA, wherein "750" represents the average molecular weight of PEG, "C" represents carbamate linker moiety, "DMA" Represents dimyristyloxypropyl. In certain embodiments, the terminal hydroxyl group of the PEG is substituted with a methyl group. One skilled in the art will readily understand that other dialkyloxypropyls can be used in the PEG-DAA conjugates of the present invention.

In addition to the above, it will be readily apparent to those skilled in the art that other hydrophilic polymers may be used instead of PEG. Examples of suitable polymers that may be used in place of PEG include polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid , And derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

In addition to the above ingredients, the lipid particles of the present invention (e. G., SNALP) may further comprise a cationic poly (ethylene glycol) (PEG) lipid or CPL Chen et al., Bioconj. Chem., 11: 433-437 (2000); U.S. Patent No. 6,852,334; PCT Publication No. WO 00/62813).

Suitable CPLs include compounds of formula (VIII)

≪ Formula (VIII)

A-W-Y

In the above formula, A, W, and Y are as described below.

In Formula (VIII), "A" is a lipid moiety that acts as a lipid anchor, such as an amphipathic lipid, a neutral lipid, or a hydrophobic lipid. Examples of suitable lipids include diacyl glycerol, dialkyl glycerol, NN-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl- It is not limited.

"W" is a polymer or oligomer, such as a hydrophilic polymer or oligomer. Preferably, the hydrophilic polymer is a biocompatible polymer that is non-immunogenic or has a low inherent immunogenicity. Alternatively, the hydrophilic polymer may be weakly antigenic when used in conjunction with an appropriate adjuvant. Suitable non-immunogenic polymers include, but are not limited to, PEG, polyamides, polylactic acid, polyglycolic acid, polylactic acid / polyglycolic acid copolymers, and combinations thereof. In a preferred embodiment, the molecular weight of the polymer is from about 250 to about 7,000 Daltons.

"Y" is a polyvalent cationic moiety. The term multivalent cationic moiety means a compound, derivative, or functional group having a positive charge, preferably at least two positive charges, at a selected pH, preferably at physiological pH. Suitable polyvalent cationic moieties include basic amino acids and derivatives thereof such as arginine, asparagine, glutamine, lysine, and histidine; Spermine; Spermidine; Cationic dendrimers; Polyamines; Polyamine sugar; And aminopolysaccharides. The polyvalent cation moiety may be linear in structure, such as linear tetra lysine, branched or dendrimer. The polyvalent cationic moiety has from about 2 to about 15 positive charges, preferably from about 2 to about 12 positive charges, more preferably from about 2 to about 8 positive charges at the selected pH value. The choice of polyvalent cationic moiety to be used can be determined by the kind of particle application required.

The charge on the polyvalent cationic moiety can be distributed around the entire particle moiety, or alternatively, can be a separate concentrate of charge density in one particular region of the particle moiety, e.g., a charge spike have. If the charge densities are distributed on the particles, the charge densities can be distributed equally or evenly. All variations of the charge distribution of the polyvalent cation moiety are encompassed by the present invention.

The lipid "A" and the non-immunogenic polymer "W " can be attached by various methods, preferably by covalent attachment. Methods known to those skilled in the art can be used for the covalent attachment of "A" and "W ". Suitable linking groups include, but are not limited to, amides, amines, carboxyls, carbonates, carbamates, esters, and hydrazone linkages. It will be apparent to those skilled in the art that "A" and "W " The reaction between two groups, one on the lipid phase and one on the polymer will provide the desired linkage. For example, if the lipid is diacylglycerol and the terminal hydroxyl is activated, for example, with NHS and DCC to form the active ester, then reacting with a polymer containing amino groups, such as polyamides (e. See U.S. Patent Nos. 6,320,017 and 6,586,559, the disclosures of which are hereby incorporated by reference in their entirety for all purposes), and an amide bond will be formed between the two groups.

In certain cases, the polyvalent cationic moiety may have an attached ligand, such as a targeting ligand, or a chelating moiety for calcium complexation. Preferably, after the ligand is attached, the cationic moiety retains a positive charge. In certain cases, the attached ligand has a positive charge. Suitable ligands include, but are not limited to, compounds or devices having reactive functional groups, such as lipids, amphipathic lipids, carrier compounds, biocompatible compounds, biomaterials, biopolymers, biomedical devices, analytically detectable compounds, The present invention also relates to a method of screening for a compound of the present invention which is capable of inhibiting the activity of a compound, enzyme, peptide, protein, antibody, immunostimulant, radioactive label, fluorescent source, biotin, drug, Or toxins.

In some embodiments, the lipid conjugate (e. G., PEG-lipid) comprises from about 0.1 mole% to about 2 mole%, from about 0.5 mole% to about 2 mole%, from about 1 mole% From about 0.6 mol% to about 1.9 mol%, from about 0.7 mol% to about 1.8 mol%, from about 0.8 mol% to about 1.7 mol%, from about 0.9 mol% to about 1.6 mol%, from about 0.9 mol% About 1.8 mole%, about 1.8 mole%, about 1 mole% to about 1.7 mole%, about 1.2 mole% to about 1.8 mole%, about 1.2 mole% to about 1.7 mole%, about 1.3 mole% 1.6 mole percent, or from about 1.4 mole percent to about 1.5 mole percent (or any range or range therein).

In another embodiment, the lipid conjugate (e. G., PEG-lipid) comprises from about 0 mole% to about 20 mole%, from about 0.5 mole% to about 20 mole%, from about 2 mole% About 20 mole percent, about 1.5 mole percent to about 18 mole percent, about 2 mole percent to about 15 mole percent, about 4 mole percent to about 15 mole percent, about 2 mole percent to about 12 mole percent, 12 mole%, or about 2 mole% (or any range or range thereof).

In a further embodiment, the lipid conjugate (e. G., PEG-lipid) comprises from about 4 mol% to about 10 mol%, from about 5 mol% to about 10 mol%, from about 5 mol% About 9 to about 9 mole percent, about 5 to about 8 mole percent, about 6 to about 9 mole percent, about 6 to about 8 mole percent, or about 5 mole percent, 6 mole percent, 7 mole percent, Mole%, 9 mole%, or 10 mole% (or any range or range thereof).

Additional proportions and ranges of lipid conjugates suitable for use in the lipid particles of the present invention are described in PCT Publication No. WO 09/127060, U.S. Pat. U.S. Patent Application Publication No. US 2011/0071208, PCT Publication No. WO2011 / 000106, and U.S. Pat. Patent application publication US 2011/0076335.

It should be understood that the ratio of the lipid conjugate (e.g., PEG-lipid) present in the lipid particles of the present invention is the target amount and that the actual amount of lipid conjugate present in the formulation may differ by, for example, +/- 2 mol% do. For example, in a 1:57 lipid particle (e.g., SNALP) formulation, the target amount of lipid conjugate is 1.4 mole%, but the actual amount of lipid conjugate is +/- 0.5 mole%, +/- 0.4 mole%, +/- 0.3 mole% %, ± 0.2 mol%, ± 0.1 mol%, or ± 0.05 mol%, where the remainder of the formulation consists of other lipid components (up to 100 mol% of the total lipid present in the particles) . Similarly, in a 7:54 lipid particle (e.g., SNALP) formulation, the target amount of lipid conjugate is 6.76 mole%, but the actual amount of lipid conjugate is +/- 2 mole%, +/- 1.5 mole%, +/- 1 mole% , ± 0.75 mol%, ± 0.5 mol%, ± 0.25 mol%, or ± 0.1 mol%, where the remainder of the formulation consists of other lipid components (up to 100 mol% of the total lipid present in the particles) .

Those skilled in the art will appreciate that the concentration of the lipid conjugate can vary depending on the rate at which the lipid conjugate and lipid particles used are likely to be fused.

By controlling the composition and concentration of the lipid conjugate, it is possible to control the rate at which the lipid conjugate is exchanged from the lipid particle, and the rate at which the lipid particle is again capable of fusion. For example, when a PEG-DAA conjugate is used as a lipid conjugate, the rate at which the lipid particles are likely to become fused can be determined by, for example, the concentration of the lipid conjugate, the molecular weight change of the PEG, or the chain length and unsaturation of the alkyl group on the PEG- As shown in FIG. Additionally, other variables, including, for example, pH, temperature, ionic strength, and the like, can be used to alter and / or control the rate at which lipid particles become fusable. Other methods that can be used to control the rate at which lipid particles are likely to fuse will be apparent to those skilled in the art from the disclosure herein. In addition, the size of lipid particles (e.g., SNALP) can be controlled by controlling the composition and concentration of the lipid conjugate.

B. Additional carrier  system

Non-limiting examples of additional lipid-based carrier systems suitable for use in the present invention include lipoflexes (see, e.g., US Patent Publication 20030203865 and Zhang et al., J. Control Release, 100: 165-180 2004)), pH-sensitive lipoplexes (see, for example, US Patent Publication 20020192275), reversibly masked lipoplexes (see, for example, US Patent Publication 20030180950), cationic lipid- (See, for example, U.S. Patent No. 6,756,054 and U.S. Patent Publication No. 20050234232), cationic liposomes (see, for example, U.S. Patent Publications 20030229040, 20020160038, and 20020012998; U.S. Patent No. 5,908,635; and PCT Publication No. WO 01/72283) (See, for example, U.S. Patent Publication No. 20030026831), pH-sensitive liposomes (see, for example, U.S. Patent Nos. 20020192274 and AU 2003210303), antibody-coated liposomes PCT (See for example WO 00/50008), cell type specific liposomes (see, for example, US Patent Publication 20030198664), liposomes containing nucleic acids and peptides (see, for example, U.S. Patent 6,207,456), derivatized with a releasable hydrophilic polymer Entrapped nucleic acids (see, for example, PCT Publication Nos. WO 03/057190 and WO 03/059322), lipid-encapsulated nucleic acids (see, for example, U.S. Patent Publication No. 20030031704), lipid- (See, for example, U.S. Patent Nos. 20030035829 and 20030072794; and U.S. Patent 6,200,599), stabilized mixtures of liposomes and emulsions (see, for example, U.S. Patent Publication No. 20030129221 and U.S. Patent No. 5,756,122), other liposomal compositions Emulsion compositions (see, for example, U.S. Patent No. 6,747,014), and nucleic acid micro-emulsions (see, for example, U.S. Patent Publication No. 20050037086).

Examples of polymer-based carrier systems suitable for use in the present invention include, but are not limited to, cationic polymer-nucleic acid complexes (i.e., polyplexes). In order to form polyplexes, nucleic acids (e. G., Interfering RNAs) are typically prepared by condensing nucleic acids with positively charged particles that interact with anionic proteoglycans at the cell surface and can be introduced into cells by endocytosis Lt; RTI ID = 0.0 > linear, branched, < / RTI > star, or dendritic polymeric structure. In some embodiments, the polyplex is a cationic polymer, such as polyethyleneimine (PEI) (see, for example, U. S. Patent No. 6,013, 240; Qbiogene as the in vivo jetPEI TM, a linear form of PEI, (PPI), polyvinylpyrrolidone (PVP), poly-L-lysine (PLL), diethylaminoethyl (DEAE) -dex (See for example, Lynn et al., J. Am. Chem. Soc. 123: 8155-8156 (2001)), chitosan, polyamidoamine (See, for example, U.S. Patent No. 6,620,805), PAMAM dendrimers (see, for example, Kukowska-Latallo et al., Proc. Natl. Acad. Sci. USA, 93: 4897-4902 ), Polyvinyl ethers (see, for example, US Patent Publication 20040156909), polycyclic amidiniums (see, for example, US Patent Publication 20030220289) Other polymers including amines, imines, guanidines, and / or imidazole groups (see, for example, US Patent 6,013,240; PCT Publication WO / 9602655; PCT Publication WO95 / 21931; Zhang et al., J. Control Release, (See for example Tiera et al., Curr. Gene Ther., 6: 59-71 (2006)) and complexes thereof with a mixture thereof RNA). In another embodiment, the polyplex comprises a cationic polymer-nucleic acid complex as described in U. S. Patent Nos. 20060211643, 20050222064, 20030125281, and 20030185890, and PCT Publication WO 03/066069; Biodegradable poly (beta -amino ester) polymer-nucleic acid complexes described in U.S. Patent Publication 20040071654; Microparticles containing a polymeric matrix as described in U.S. Patent Publication 20040142475; Other microparticulate compositions as described in U.S. Patent Publication 20030157030; A condensed nucleic acid complex as described in U. S. Patent Publication 20050123600; And the nanocapsule and microencapsulation compositions described in AU 2002358514 and PCT Publication WO 02/096551.

In certain cases, the interfering RNA can be complexed with a cyclodextrin or a polymer thereof. Non-limiting examples of cyclodextrin-based carrier systems include the cyclodextrin-modified polymer-nucleic acid complexes described in U.S. Patent Publication 20040087024; Linear cyclodextrin copolymer-nucleic acid complexes described in U.S. Patent Nos. 6,509,323, 6,884,789, and 7,091,192; And the cyclodextrin polymer-complexing agent-nucleic acid complexes described in U.S. Patent No. 7,018,609. In certain other examples, the interfering RNA can be complexed to a peptide or polypeptide. Examples of protein-based carrier systems include, but are not limited to, the cationic oligopeptide-nucleic acid complexes described in PCT Publication No. WO95 / 21931.

VI . Preparation of lipid particles

SNALPs in which lipid particles of the invention, e.g., nucleic acids, such as interfering RNA (e.g., siRNA), are trapped within the lipid portion of the particle and are protected from degradation, can be used in a continuous mixing method, -line) < / RTI > dilution process, as described herein.

In certain embodiments, the cationic lipids may comprise lipids of formula I-III, or salts thereof, alone or in combination with other cationic lipids. In another embodiment, the non-cationic lipid is selected from the group consisting of egg sphingomyelin (ESM), distearoylphosphatidylcholine (DSPC), dioloylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (DPPC), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, 14: 0 PE (1,2-dimyristoyl-phosphatidylethanolamine (DMPE)), 16: Phosphatidylethanolamine (DPPE)), 18: 0 PE (1,2-distearoyl-phosphatidylethanolamine (DSPE)), 18: 1 PE (1,2-diolureo (DOPE), 18: 1 trans PE (1,2-dielaidoyl-phosphatidylethanolamine (DEPE)), 18: 0-18: 1 PE (1-stearoyl- Oleophilic-ethanolamine (POPE), polyethylene glycol-based polymers (e.g., PEG 2000, PEG 2000, PEG 2000, PEG 5000, PEG-modified Diacylglycerol, or PEG-modified dialkyloxypropyl), cholesterol, derivatives thereof, or combinations thereof.

In certain embodiments, the invention provides a continuous mixing method, such as providing an aqueous solution comprising a nucleic acid (e. G., Interfering RNA) in a first reservoir, providing an organic lipid solution in a second reservoir, wherein , The lipid present in the organic lipid solution is solubilized in an organic solvent, such as a lower alkanol, such as ethanol, a lipid vesicle (e.g., liposome) encapsulating the nucleic acid in a lipid vesicle, Lipid particles (e.g., SNALP) produced through a process comprising mixing an aqueous solution with an organic lipid solution such that the lipid solution is mixed with the aqueous solution for production. The process and apparatus for carrying out this process are described in detail in U.S. Patent Publication 20040142025, the full text of which is incorporated herein by reference in its entirety.

When the lipid and buffer solution is continuously introduced into the mixing environment, such as into the mixing chamber, the lipid solution and the buffer solution are continuously diluted, thereby producing a lipid vesicle substantially immediately upon mixing. As used herein, the phrase "continuous dilution of the lipid solution and the buffer solution" (and variants) generally means that the lipid solution is diluted sufficiently fast in the hydration process with sufficient force to achieve vesicle formation. By mixing an aqueous solution comprising the nucleic acid with an organic lipid solution, the organic lipid solution is subjected to successive stepwise dilutions in the presence of a buffer solution (i.e., aqueous solution) to produce nucleic acid-lipid particles.

The nucleic acid-lipid particles formed using the continuous mixing method generally have a pH of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm From about 70 nm to about 90 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 nm to about 90 nm, from about 80 nm to about 90 nm, nm, or about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, Its arbitrary ratio or the range within it). The particles thus formed do not agglomerate and are sized by size to achieve an arbitrary uniform particle size.

In another embodiment, the present invention relates to a direct dilution process comprising forming a lipid vesicle (e. G., Liposome) solution and immediately introducing the lipid vesicle solution into a collection container containing a direct controlled amount of dilution buffer (E. G., SNALP) that is produced via < / RTI > In a preferred aspect, the collection vessel comprises one or more elements formed to agitate the contents of the collection vessel to facilitate dilution. In one aspect, the amount of diluent buffer present in the collection vessel is substantially equal to the volume of the lipid vesicle solution introduced. As a non-limiting example, a lipid vesicle solution in 45% ethanol will advantageously produce smaller particles when introduced into a collection vessel containing the same volume of diluent buffer.

In another embodiment, the present invention provides nucleic acid-lipid particles (e. G., SNALP) produced via an in-line dilution process in which a third reservoir containing a diluent buffer is fluidly connected to a second mixing region. In this embodiment, the lipid vesicle (e. G., Liposome) solution formed in the first mixing region is immediately mixed directly with the dilution buffer in the second mixing region. In a preferred aspect, the second mixing zone comprises a lipid vesicle solution and a T-linker arranged such that the dilution buffer flow is matched as a 180 ° opposite flow; A connector that provides a smaller angle, e. G., From about 27 degrees to about 180 degrees (e. G., About 90 degrees) may be used. The pump mechanism delivers the buffer to the second mixing zone in a controllable flow. In one aspect, the flow rate of the diluent buffer provided in the second mixing zone is controlled to be substantially equal to the flow rate of the lipid vesicle solution introduced therefrom from the first mixing zone. This embodiment advantageously permits greater control over the flow of dilution buffer that mixes with the lipid vesicle solution in the second mixing zone and thus the concentration of the lipid vesicle solution in the buffer throughout the second mixing process. Control of the diluent buffer flow rate allows the formation of small particle sizes at advantageously reduced concentrations.

Apparatus for carrying out the process and the direct dilution and in-line dilution process is described in detail in U. S. Patent Publication 20070042031, the full text of which is incorporated herein by reference in its entirety.

Generally, the nucleic acid-lipid particles formed using the direct dilution and in-line dilution process have a pH of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm , About 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, nm or about 80 nm or about 120 nm, 110 nm, 100 nm, 90 nm or 80 nm or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, nm < / RTI > (or any proportion or range thereof). The particles thus formed do not agglomerate and are sized by size to achieve an arbitrary uniform particle size.

If desired, the lipid particles of the present invention (e. G., SNALP) can be sized by any method available for size classification of the liposomes. Classification by size can be performed to achieve a relatively narrow distribution of the desired size range and particle size.

Several techniques are available to classify particles into desired sizes. Classification methods for size as used for liposomes and equally applicable to the particles of the present invention are described in U.S. Patent 4,737,323, the full text of which is incorporated herein by reference in its entirety. Ultrasonic treatment of the particle suspension by bath or probe ultrasonication progressively reduces the size to particles less than about 50 nm in size. Homogenization is another method that relies on shear energy to break larger particles into smaller particles. In a typical homogenization procedure, the particles are recycled through a standard emulsion homogenizer until a selected particle size, typically about 60 to about 80 nm, is observed. In both methods, the particle size distribution can be monitored by conventional laser beam particle size identification, or QELS.

Extrusion of particles through small pore polycarbonate membranes or asymmetric ceramic membranes is also an effective method for reducing particle size to relatively well-defined size distributions. Generally, the suspension is cycled through the membrane one or more times until the desired particle size distribution is achieved. The particles can be extruded continuously through the membrane of smaller pores to achieve a gradual reduction in size.

In some embodiments, the nucleic acid present in the particle is pre-condensed, for example, as described in U. S. Patent Application Serial No. 09 / 744,103, the disclosure of which is incorporated herein by reference in its entirety.

In another embodiment, the method may further comprise adding a non-lipid polyvalent cation useful for performing lipofection of cells using the composition of the present invention. Suitable Examples of non-cationic lipid is a polyhydric hexamethylene di meth Lin bromide (trade name polybrene (POLYBRENE) ^® under the Aldrich Chemical Company (Aldrich Chemical Co., sold by Milwaukee, Wisconsin, USA)), or another salt of hexamethylene di meth Lin . Other suitable multivalent cations include, for example, salts of poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine, polyallylamine, and polyethyleneimine. The addition of these salts is preferably carried out after the particles are formed.

In some embodiments, the nucleic acid to lipid ratio (mass / mass ratio) in the formed nucleic acid-lipid particles (e.g., SNALP) ranges from about 0.01 to about 0.2, from about 0.05 to about 0.2, from about 0.02 to about 0.1, 0.1, or about 0.01 to about 0.08. The ratio of the starting material (input) is also within the above range. In another embodiment, the particle formulation comprises a nucleic acid to lipid mass ratio (corresponding to 1.25 mg total lipid / 50 μg nucleic acid) of about 400 μg nucleic acid / 10 mg total lipid or about 0.01 to about 0.08, more preferably about 0.04 . In another preferred embodiment, the particles have a nucleic acid: lipid mass ratio of about 0.08.

In another embodiment, the lipid to nucleic acid ratio (mass / mass ratio) in the formed nucleic acid-lipid particles (e.g., SNALP) ranges from about 1 (1: 1) to about 100 (100: ) To about 100 (100: 1), from about 1: 1 to about 50: 1, from about 2: 1 to about 50: 1, from about 3: (50: 1), about 4 (4: 1) to about 50 (50: 1), about 5 (5: 1) to about 50 (25: 1) to about 25 (25: 1) to about 25 (25: 1) About 5 (5: 1) to about 25 (25: 1), about 5 (5: 1) to about 20 ), About 5 (5: 1) to about 10 (10: 1), or about 5 (5: 1), 6 (6: 1), 7 15 (15: 1), 16 (16: 1), 10 (10: 1), 10 23 (23: 1), 17 (17: 1), 18 (18: 1), 19 1), 24 (24: 1), or 25 (25: 1), or any range or range thereof. The ratio of the starting material (input) is also within the above range.

As discussed previously, lipids may further include CPL. Various general methods for the production of SNALP-CPL (CPL-containing SNALP) are discussed herein. Two common techniques include "post-insertion" techniques, such as insertion of CPL into pre-formed SNALPs, and "standard" techniques in which the CPL is included in the lipid mixture, for example, during the SNALP formation step. The post-insertion technique produces SNALPs with CPL primarily on the outer surface of the SNALP bilayer membrane, while the standard technique provides a SNALP with CPL on both the inner and outer surfaces. The method is particularly useful for an endoplasmic reticulum made of a phospholipid (which may contain cholesterol) and an endoplasmic reticulum containing a PEG-lipid (such as PEG-DAA and PEG-DAG). Methods for making SNALP-CPL are described, for example, in U.S. Patent Nos. 5,705,385, which is incorporated herein by reference in its entirety for all purposes; 6,586,410; 5,981,501; 6,534,484; And 6,852,334; U.S. Patent Application Publication 20020072121; And PCT Publication WO 00/62813.

VII . Kit

The present invention also provides kit-shaped lipid particles (e. G., SNALP). In some embodiments, the kit comprises a container that is compartmentalized to retain various components of the lipid particle (e.g., active agent or therapeutic agent, such as nucleic acid and individual lipid components of the particles). Preferably, the kit comprises a container (e. G., A vial or ampoule) having lipid particles of the invention (e. G., SNALP) produced by one of the methods presented herein. In certain embodiments, the kit may further comprise an endosomal membrane destabilizing agent (e.g., a calcium ion). The kits generally contain the particle compositions of the present invention as a suspension in a pharmaceutically acceptable carrier or in dehydrated form, together with instructions for their rehydration (if lyophilized) and administration.

SNALP agents of the invention can be tailored to preferentially target certain cells, tissues, or organs of interest. Preferential targeting of SNALP can be accomplished by controlling the composition of SNALP itself. In certain embodiments, the kits of the invention comprise these lipid particles, wherein the particles are present in the container as a suspension or in dehydrated form.

In certain cases, the attachment of the targeting moiety to the surface of the lipid particles may be desirable to further enhance the targeting of the particles. Methods of attaching targeting moieties (e. G., Antibodies, proteins, etc.) to lipids (e. G., Those used in the particles of the invention) are known to those skilled in the art.

VIII . Administration of lipid particles

Once formed, the lipid particles (e. G., SNALP) of the present invention are particularly useful for the introduction of nucleic acids (e. G., Interfering RNA, e. Thus, the present invention also provides a method for introducing a nucleic acid (e. G., An interfering RNA) into a cell. In certain embodiments, the nucleic acid (e. G., Interfering RNA) can be an infected cell such as reticuloendothelial cells (e. G., Macrophages, mononuclear cells, etc.) and fibroblasts, endothelial cells ), And / or platelet cells. The method may be performed by first forming particles in vitro or in vivo as described above, and then contacting the particles with the cells for a time sufficient for the interfering RNA to transfer to the cells.

The lipid particles of the present invention (e. G., SNALP) can be adsorbed to almost any cell type in which they are mixed or contacted. Once adsorbed, the particles may be intracellularly transferred by a portion of the cell, the lipid may be exchanged with the cell membrane, or may be fused with the cell. Delivery or integration of the nucleic acid (e. G., Interfering RNA) portion of the particle can occur through any one of these pathways. In particular, when fusion occurs, the particle film is incorporated into the cell membrane, and the contents of the particles are combined with the intracellular fluid.

The lipid particles of the present invention (e. G., SNALP) may be administered alone or in admixture with a pharmaceutically acceptable carrier (e. G., Physiological saline or phosphate buffer) selected according to the route of administration and standard pharmaceutical practice. In general, conventional buffered saline (e.g., 135-150 mM NaCl) will be used as a pharmaceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, 0.4% saline, 0.3% glycine and the like, for example, glycoproteins for improving stability such as albumin, lipoprotein, globulin and the like. Additional suitable carriers are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids and the like. The phrase "pharmaceutically acceptable" means molecular entities and compositions that do not produce an allergic or similar adverse reaction when administered to a human.

A pharmaceutically acceptable carrier is generally added after lipid particle formation. Thus, after the formation of lipid particles (e.g., SNALP), the particles may be diluted in a pharmaceutically acceptable carrier, such as conventional buffered saline.

The concentration of the particles in the pharmaceutical preparation may vary widely, i.e., less than about 0.05%, generally at least about 2 to 5% to about 10 to 90% by weight, and depending on the particular mode of administration chosen, Will be selected. For example, the concentration can be reduced to lower the fluid load associated with the treatment. This may be particularly desirable in patients with atherosclerosis-associated congestive heart failure or severe hypertension. Alternatively, particles of irritating lipids can be diluted to a low concentration to alleviate inflammation at the site of administration.

The pharmaceutical compositions of the present invention can be sterilized by conventional, well known sterilization techniques. The aqueous solution may be packaged for use or filtered under aseptic conditions and lyophilized, and the lyophilized preparation is combined with the sterile aqueous solution prior to administration. The composition may contain pharmaceutically acceptable auxiliary substances necessary to bring it close to the physiological conditions, such as pH adjusting and buffering agents, wall thickening agents and the like, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride. In addition, particle suspensions may contain lipid-protecting agents that protect lipids against free radicals and lipid-peroxidative damage during storage. A lipophilic free radical quencher such as alpha tocopherol, and a water soluble iron-specific chelator such as a peroxomethane.

In some embodiments, the lipid particles (e. G., SNALP) of the present invention are particularly useful in methods for the therapeutic delivery of one or more nucleic acids comprising interfering RNA sequences (e. G., SiRNA). In particular, it is an object of the present invention to provide a method of treating human alcoholic intoxication in vivo by downregulating or silencing the transcription and / or translation of one or more ALDH isoenzymes.

A. In vivo  administration

Systemic delivery for in vivo therapy, such as delivery of the therapeutic nucleic acid to a remote target cell via, for example, circulation, may be accomplished using nucleic acid-lipid particles, such as those disclosed in PCT Publication WO 05/007196, WO 05/121348, WO 05/120152, and WO 04/002453. The present invention also provides fully encapsulated lipid particles suitable for repeated administration, non-immunogenic, small size, and to protect nucleic acids from neclease breakdown in serum.

For in vivo administration, administration can be by any means known in the art, for example, by injection, oral administration, inhalation (e.g. intranasal or intramuscular), transdermal application, or rectal administration . The administration can be carried out in single or divided doses. The pharmaceutical composition may be administered parenterally, i. E. Intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In some embodiments, the pharmaceutical composition can be administered intravenously or intraperitoneally by bolus injection (see, for example, U.S. Patent 5,286,634). Intracellular nucleic acid transfer is also described in Straubringer et al., Methods Enzymol., 101: 512 (1983); [Mannino et al., Biotechniques, 6: 682 (1988); [Nicolau et al., Crit. Rev. Ther. Drug Carrier Syst., 6: 239 (1989); And [Behr, Acc. Chem. Res., 26: 274 (1993). Another method of administering lipid-based therapeutics is described, for example, in U.S. Patent 3,993,754; 4,145,410; 4,235,871; 4,224,179; 4,522,803; And 4,588,578. Lipid particles can be administered by direct injection at the disease site or by injection at a site remote from the disease site (see, for example, Culver, HUMAN GENE THERAPY, MaryAn Liebert, Inc., Publishers, New York. pp. 70-71 (1994)). The above references are incorporated herein by reference in their entirety for all purposes.

In embodiments where the lipid particles of the present invention (e.g., SNALP) are administered intravenously, at least about 5%, 10%, 15%, 20%, or 25% 12, 24, 36, or 48 hours. In another embodiment, a large proportion of about 20%, 30%, 40% and about 60%, 70% or 80% of the total injected dose of lipid particles is injected at about 8, 12, 24, 36, And later in the plasma. In certain instances, greater than about 10% of the plurality of particles are present in the plasma of the mammal after about 1 hour of administration. In certain other examples, the presence of lipid particles is detectable at least about 1 hour after administration of the particles. In some embodiments, the presence of the therapeutic nucleic acid, such as an interfering RNA molecule, is detectable in the cells after about 8, 12, 24, 36, 48, 60, 72 or 96 hours of administration. In another embodiment, down-regulation of the expression of a target sequence, such as a virus or host sequence, by an interfering RNA (e.g., siRNA) is detected after about 8, 12, 24, 36, 48, 60, 72, It is possible. In another embodiment, down-regulation of expression of a target sequence, such as a virus or host sequence, by an interfering RNA (e. G., SiRNA) occurs preferentially in infected and / or infectable cells. In a further embodiment, the presence or effect of intracellular interfering RNA (e.g., siRNA) at or near the site of administration may occur after about 12, 24, 48, 72, or 96 hours of administration, or about 6, 8 , 10, 12, 14, 16, 18, 19, 20, 22, 24, 26, or 28 days. In a further embodiment, the lipid particles of the invention (e. G., SNALP) are administered parenterally or intraperitoneally.

The compositions of the present invention may be prepared in an aerosol formulation (i. E., They may be "sprayed") administered alone or in combination with other suitable ingredients via inhalation (e.g., intramuscularly or intramuscularly) et al., Am. J. Sci., 298: 278 (1989)). Aerosol formulations may be introduced into an acceptable compressed propellant, such as dichlorodifluoromethane, propane, nitrogen, and the like.

In certain embodiments, the pharmaceutical composition may be delivered by intranasal spray, inhalation, and / or other aerosol delivery vehicles. Methods of delivering nucleic acid compositions directly to the lungs via intranasal aerosol spray are described, for example, in U.S. Patent Nos. 5,756,353 and 5,804,212. Similarly, delivery of drugs using intramolecular microparticle resins and lyophosphatidyl-glycerol compounds (US Patent 5,725,871) is also well known in the pharmaceutical industry. Similarly, transmucosal drug delivery in the form of a polytetrafluoroethylene support matrix is described in U.S. Patent 5,780,045. The disclosure of which is incorporated herein by reference in its entirety for all purposes.

For example, formulations suitable for parenteral administration, such as by intraarticular (intraarticular), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, may be formulated to contain an antioxidant, a buffering agent, a bacteriostatic agent, Aqueous and non-aqueous, isotonic sterile injection solutions which may contain solutes which render the skin isotonic with the blood of the woman, and aqueous and non-aqueous sterile injection solutions which may include suspending agents, solubilizers, thickening agents, stabilizers, Aqueous sterile suspensions. In practicing the present invention, the composition is preferably administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intracystically, or intravaginally.

Generally, when administered intravenously, the lipid particle formulation is formulated with a suitable pharmaceutical carrier. Many pharmaceutically acceptable carriers can be used in the compositions and methods of the present invention. Suitable formulations for use in the present invention are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). Various aqueous carriers such as water, buffered water, 0.4% saline, 0.3% glycine and the like may be used and may include glycoproteins such as albumin, lipoprotein, globulin and the like for improving stability. In general, conventional buffered saline (135-150 mM NaCl) will be used as the pharmaceutically acceptable carrier, but other suitable carriers will be sufficient. These compositions can be sterilized by conventional liposomal sterilization techniques, such as filtration. The compositions may also contain pharmaceutically acceptable auxiliary substances necessary for making them close to the physiological conditions such as pH adjusting and buffering agents, thickening agents, wetting agents and the like, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride and calcium chloride, sorbitan mono Laurate, triethanolamine oleate, and the like. The composition may be sterilized using the techniques described above, or alternatively, may be produced under sterile conditions. The resulting aqueous solution may be packaged for use or filtered under aseptic conditions and lyophilized, and the lyophilized preparation is combined with a sterile aqueous solution prior to administration.

In certain applications, the lipid particles disclosed herein may be delivered to an individual via oral administration. Particles are included with excipients and may be used in the form of ingestible tablets, buccal tablets, troches, capsules, pills, lozenges, elixirs, mouthwashes, suspensions, oral sprays, syrups, wafers, and the like See, for example, U.S. Patent Nos. 5,641,515, 5,580,579, and 5,792,451, the disclosures of which are hereby incorporated herein by reference in their entirety for all purposes). These oral dosage forms may also contain: binders, gelatin; Excipients, lubricants, and / or flavors. When the unit dosage form is a capsule, it may contain, in addition to the above-described materials, a liquid carrier. A variety of different materials may be present as coatings or to otherwise modify the physical form of the dosage unit. Of course, any substance used in the manufacture of any unit dosage form should be pharmaceutically pure and substantially non-toxic in the amounts used.

In general, these oral preparations may contain at least about 0.1% or more of the lipid particles, but the proportion of the particles may of course vary and conveniently can be about 1% or 2% of the weight or volume of the total formulation About 60% or 70% or more. Thus, the amount of particles in each therapeutically useful composition may be prepared in such a manner that a suitable dosage amount is obtained at any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life and other pharmacological considerations will be considered by those skilled in the art of making such pharmaceutical agents, and accordingly, various dosages and therapeutic regimens may be preferred have.

Suitable formulations for oral administration may comprise: (a) an effective amount of a packaged therapeutic nucleic acid (e. G., Interfering RNA) suspended in a liquid solution, such as a diluent such as water, saline, or PEG 400; (b) a capsule, sachet, or tablet containing a predetermined amount of therapeutic nucleic acid (e.g., interfering RNA) as a liquid, solid, granule, or gelatin, respectively; (c) a suspension in a suitable liquid; And (d) a suitable emulsion. Tablet forms may include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid and other excipients, , Binders, diluents, buffers, moisturizers, preservatives, flavors, dyes, disintegrants, and pharmaceutically acceptable carriers. The lozenge forms include flavoring agents, such as therapeutic nucleic acids (e. G., Interfering RNA) in sucrose, and pastilles or sucrose and therapeutic nucleic acids containing the therapeutic nucleic acid in an inert base such as gelatin and glycerin Acacia emulsions, gels, and the like, containing carriers known in the art.

In another example of their use, the lipid particles may be incorporated into a wide range of topical dosage forms. For example, suspensions containing nucleic acid-lipid particles, such as SNALP, may be formulated and administered as a gel, oil, emulsion, topical cream, paste, ointment, lotion, foam, mousse,

In preparing pharmaceutical formulations of the lipid particles of the present invention, it is preferred to use large amounts of purified particles to reduce or eliminate particles having a therapeutic agent, such as a nucleic acid, bound to the particle or outer surface.

The methods of the invention can be practiced in a variety of hosts. Preferred hosts include mammalian species such as primates (e.g., humans and chimpanzees and other nonhuman primates), dogs, cats, horses, cows, sheep, goats, rodents (e.g. rats and mice), rabbits, .

The amount of the administered particles will be determined by the ratio of the therapeutic nucleic acid (e. G., Interfering RNA) to lipid, the particular therapeutic nucleic acid used, the disease or disorder to be treated, the age, weight and condition of the patient, (E.g., body weight), preferably about 0.1 to about 5 mg / kg (body weight), or about 10 ^{8-10 10} particles / dose (e.g., injection).

B. In vitro  administration

For in vitro application, the delivery of the therapeutic nucleic acid (e. G., Interfering RNA) can be by propagation into a plant or animal origin, a vertebrate or invertebrate animal, and any cell or tissue of any species, grown in culture . In a preferred embodiment, the cell is an animal cell, more preferably a mammalian cell, most preferably a human cell.

Contact between the cell and lipid particles occurs in a biologically compatible medium when performed in vitro. The concentration of the particles varies widely depending on the particular application, but is generally about 1 [mu] mol to about 10 mmol. Treatment of cells with lipid particles is generally carried out at physiological temperature (about 37 DEG C) for about 1 to 48 hours, preferably about 2 to 4 hours.

In one group of preferred embodiments, the lipid particles of the suspension is from about 10 ³ to about 10 ⁵ cells / ml, preferably from about 2 x 10 ⁴ cells / ml cell density 60-80% fusion also (confluent) having a more Is added to the plated cells. The concentration of the suspension added to the cells is preferably about 0.01 to 0.2 占 퐂 / ml, more preferably about 0.1 占 퐂 / ml.

To the extent that tissue culture of the cells may be necessary, this is well known in the art. See, for example, Freshney, Culture of Animal Cells, a Manual of Basic Technique, 3rd Ed., Wiley-Liss, New York (1994), Kuchler et al., Biochemical Methods in Cell Culture and Virology, Hutchinson and Ross, Inc. (1977)], and references cited therein, provide general guidance on cell culture. The cultured cell system will often be in the form of a single layer of cells, but cell suspensions are also used.

Endosomal release parameter (ERP) assays can be used to optimize the delivery efficiency of the SNALPs or other lipid particles of the present invention. The ERP assay is described in detail in U. S. Patent Publication 20030077829, which is incorporated herein by reference in its entirety for all purposes. More particularly, the purpose of the ERP assay is to determine the effect of various cationic lipid and helper lipid components of SNALP or other lipid particles on the basis of their relative effects on binding / absorption or fusion / destabilization with endosomal membranes . By this assay, the extent to which each component of SNALP or other lipid particles affects the delivery efficiency and thereby optimizes SNALP or other lipid particles can be determined quantitatively. Generally, ERP assays measure the expression of reporter proteins (e.g., luciferase, beta -galactosidase, green fluorescent protein (GFP), etc.) and, in some instances, SNALP preparations optimized for expression plasmids It would also be appropriate to encapsulate the interfering RNA. In another example, an ERP assay can be tailored to measure downregulation of transcription or translation of a target sequence in the presence or absence of an interfering RNA (e. G., SiRNA). By comparing ERPs for each of the various SNALPs or other lipid particles, it is possible to easily determine an optimized system, for example, SNALP or other lipid particles exhibiting maximum absorption in cells.

C. Cells for delivery of lipid particles

The compositions and methods of the present invention are particularly well suited for treating alcoholism by optimizing ALDH gene expression in vivo. The present invention provides a method of treating a mammal such as a dog, a cat, a horse, a cow, a sheep, a goat, a rodent (e.g., a mouse, a rat and a guinea pig), a rabbit, Lt; RTI ID = 0.0 > vertebrate < / RTI > species.

D. Detection of Lipid Particles

In some embodiments, the lipid particles (e.g., SNALP) of the present invention are detectable within about 1, 2, 3, 4, 5, 6, 7, 8 hours or more times in a subject. In another embodiment, the lipid particles of the invention (e. G., SNALP) are administered at a dosage of about 8, 12, 24, 48, 60, 72, or 96 hours, or about 6,8, 10,12 , 14, 16, 18, 19, 22, 24, 25, or 28 days. The presence of the particles can be detected in the cells, tissues, or other biological samples from the subject. Particles may be detected, for example, by direct detection of the particles, detection of the therapeutic nucleic acid, such as an interfering RNA (e.g., siRNA) sequence, detection of the target sequence of interest (i. E., By detection of expression or reduced expression of the sequence of interest) , Detection of a compound modulated by an EBOV protein (e. G., Interferon), detection of viral load in a subject, or a combination thereof.

1. Particle detection

The lipid particles of the present invention, such as SNALP, can be detected using any method known in the art. For example, the label can be attached to the component of the lipid particle directly or indirectly using methods well known in the art. A wide variety of labels may be used and labels are selected according to the required sensitivity, ease of conjugation with lipid particle components, stability requirements, and available instrumentation and processing regulations. Suitable labels include, but are not limited to, fluorescent labels such as fluorescent dyes (e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon Green (TM); rhodamine and derivatives such as Texas Red radioisotope such as ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C, ³² P (red), tetraiodine isothiocyanate (TRITC), digoxigenin, biotin, picoeritrin, AMCA, CyDyes , ³³ P, etc. Enzymes such as horseradish peroxidase, alkaline phosphatase, etc .; spectroscopic colorimetric labels such as colloidal gold or colored glass or plastic beads such as polystyrene, polypropylene, latex and the like, The label can be detected using any means known in the art.

2. Detection of nucleic acid

The nucleic acid (e. G., Interfering RNA) is detected and quantified herein by any of a number of means well known to those skilled in the art. Detection of nucleic acids can be carried out by well known methods such as Southern analysis, Northern analysis, gel electrophoresis, PCR, radioactive labeling, scintillation counting, and affinity chromatography. Additional analytical biochemical methods such as spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography may also be used.

The choice of nucleic acid hybridization approach is not important. A variety of nucleic acid hybridization schemes are known to those skilled in the art. For example, conventional methods include sandwich validation and competitive or displacement validation. Hybridization techniques are described, for example, in Nucleic Acid Hybridization, A Practical Approach, Eds. Hames and Higgins, IRL Press (1985).

The sensitivity of the hybridization assay can be improved through the use of a nucleic acid amplification system that increases the number of target nucleic acids to be detected. In vitro amplification techniques are known which are suitable for the generation of nucleic acid fragments for sequential amplification or subsequent subcloning for use as molecular probes. (Such as, for example, PCR amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ-replicase amplification, and other RNA polymerase mediated techniques Examples of techniques that are sufficient to be described are described in Sambrook et al., In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2000); And Ausubel et al., SHORT PROTOCOLS IN MOLECULAR BIOLOGY, eds., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (2002); And U.S. Patent 4,683,202; [PCR Protocols, A Guide to Methods and Applications (Innis et al. Eds.) Academic Press Inc. San Diego, CA (1990); [Arnheim & Levinson (October 1, 1990), C & [The Journal of NIH Research, 3: 81 (1991)]; [Kwoh et al., Proc. Natl. Acad. Sci. USA, 86: 1173 (1989); [Guatelli et al., Proc. Natl. Acad. Sci. USA, 87: 1874 (1990); [Lomell et al., J. Clin. Chem., 35: 1826 (1989); [Landegren et al., Science, 241: 1077 (1988)]; [Van Brunt, Biotechnology, 8: 291 (1990)]; [Wu and Wallace, Gene, 4: 560 (1989)]; [Barringer et al., Gene, 89: 117 (1990)]; And Sooknanan and Malek, Biotechnology, 13: 563 (1995). An improved method of cloning amplified nucleic acids in vitro is described in U.S. Patent No. 5,426,039. Other methods described in the art are nucleic acid sequence based amplification (NASBA (TM), Cangene, Mississaugo, Ontario) and Q-replicase systems. These systems designed to extend or lyse PCR or LCR primers only when the selected sequence is present can be used directly to identify mutants. Alternatively, the selected sequence may be amplified, for example, using, for example, a nonspecific PCR primer, and the amplified target region may be probed for a specific sequence that represents a subsequent mutation. The above references are incorporated herein by reference in their entirety for all purposes.

For example, nucleic acid for use as a gene probe or as an inhibitor component for use as a probe in an in vitro amplification method is generally described, for example, in Needham VanDevanter et al., Nucleic Acids Res., 12: 6159 (1984) , According to the solid phase phosphoramidite triester method described in Beaucage et al., Tetrahedron Letts., 22: 1859 1862 (1981), using an automated synthesizer as described in US Pat. If necessary, purification of the polynucleotide is generally carried out by natural acrylamide gel electrophoresis or anion exchange HPLC as described in Pearson et al., J. Chrom., 255: 137 149 (1983). The sequence of the synthetic polynucleotides can be confirmed using the chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology, 65: 499.

A separate means for determining transcription levels is in situ hybridization. In situ hybridization assays are well known and are generally described in Angerer et al., Methods Enzymol, 152: 649 (1987). In in situ hybridization assays, the cells are fixed on a solid support, typically a glass slide. When DNA is to be probed, the cells are denatured with heat or alkaline. The cell is then contacted with the hybridization solution at a temperature that allows annealing of the labeled specific probe. The probe is preferably labeled with a radioactive isotope or fluorescent reporter.

IX . Example

The present invention will be described in more detail by way of specific examples. The following examples are provided for illustrative purposes and are not intended to limit the invention in any way. Those skilled in the art will readily recognize a variety of non-critical parameters that may be altered or modified to achieve essentially the same result.

Example  One.

This example demonstrates that serum-stable nucleic acid-lipid particles (SNALPs) containing siRNA targeting ALDH2 reduce aldehyde dehydrogenase second gene expression in murine hepatocellular cell lines in vitro.

matter:

All siRNA molecules used in this study were chemically synthesized and annealed using standard procedures.

The aldehyde dehydrogenase 2 (ALDH2) -targeted siRNA sequence used in this study:

Here, 'r' represents a ribonucleotide and 'm' represents a 2'-O-methylated ribonucleotide, and the lack of these prefixes represents a deoxynucleotide.

In addition, non-targeted siRNA was included as a control in the study. The siRNA targets the firefly luciferase gene and is not intended to have any specific gene silencing activity in mammalian cells.

Way:

The nucleic acid-lipid particles consisted of the following "1:57" formulation: 1.4% PEG2000-C-DMA; 57.1% DLinDMA; 7.1% DPPC; And 34.3% cholesterol. Generally, in a 1:57 formulation, the amount of DLinDMA was 57.1 mole% ± 5 mole%, the amount of lipid conjugate was 1.4 mole% ± 0.5 mole%, and the remainder of 1:57 formulation was a non-cationic lipid , Phospholipids, cholesterol, or two mixtures) and the final siRNA / lipid ratio was about 0.11 to 0.15 (wt / wt). Upon formation of the siRNA-loaded particles, the average particle size was 85-100 nm in diameter.

Mammalian cell treatment and mRNA  analysis:

Primary hepatocytes were isolated from mice and maintained as adherent cultures in 96-well plates. ALDH-2-targeted or non-targeted control group SNALP was added to the culture medium at an siRNA concentration of 0.03125 to 0.5 [mu] g / mL; Each treatment condition was performed in triplicate wells. After incubation for about 24 h in the presence of SNALP, cells were harvested and lysed for mRNA analysis. Mouse ALDH2 mRNA in the cell lysate was normalized to the branched DNA assay (quantization tijen ^®, Bahia matrix) using the first measurement, and mouse glyceraldehyde 3-phosphate dehydrogenase with formaldehyde to (GAPDH).

result:

Table A shows the gene silencing activity of lipid-nucleic acid treatment (compared to the average of 9 replicate test wells, ± 6.9% standard deviation) of GAPDH-normalized ALDH2 mRNA measured in untreated culture wells set to "100.0% Average of triplet wells, ± standard deviation).

<Table A>

conclusion:

All four siRNA preparations targeting mouse ALDH2 showed gene silencing activity in a dose-response manner in isolated primary mouse hepatic cells. Among the four tested strains, the duplex 1 showed the highest activity and the duplex 4 showed the least activity.

Example 2

This example shows that SNALP containing siRNA targeting ALDH2 reduces aldehyde dehydrogenase second gene expression in whole animal systems via the intravenous route of administration.

matter:

The murine ALDH-2 siRNA duplex 1 and the non-targeted control group used in this study are described in Example 1.

Way:

The SNALP formulation was prepared as described in Example 1.

Animal processing:

Female BALB / c mice were obtained from Harlan Labs. Animals were administered a single dose of SNALP-formulated siRNA, or phosphate-buffered saline, via a 10 mL / kg intravenous injection in the side-tail vein. The administered siRNA dose was 0.025, 0.050 or 0.25 mg / kg (body weight). SNALP after injection about 48 h, the liver was collected euthanized animal, and RNA within the concentrator (RNAlater) ^® RNA stabilizing solution.

Organizational Analysis:

A, (a subsidiary of the present Bahia matrix wave nomikseu (Panomics), California, USA, Fremont): [494. Judge et al , 2006, Molecular Therapy 13] Quorn tijen ^® branched DNA test as described essentially by reference to the liver tissue And analyzed for normalized mouse ALDH2 mRNA levels for GAPDH mRNA levels.

result:

Table B summarizes the gene silencing activity of SNALP treatment compared to the amount of GAPDH-normalized ALDH2 mRNA (mean of 6 animals, ± 6.3% standard deviation) measured in control PBS-treated animals, optionally set to "100.0% Mean, ± standard deviation of horses).

<Table B>

conclusion:

Single intravenous administration of SNALP-formulated siRNA 1 targeting mouse ALDH2 produced the ALDH2 gene silencing activity in a dose-response manner in mice.

Example  3.

This example illustrates a method for preparing serum-stable nucleic acid-lipid particles of the present invention.

siRNA molecules are chemically synthesized and annealed using standard procedures.

In some embodiments, the siRNA molecule is encapsulated into a serum-stable nucleic acid-lipid particle consisting of the following lipids: (1) lipid conjugate PEG2000-C-DMA (3-N - [(methoxypoly (ethylene glycol) 2000) carbamoyl] -1, 2-dimyristyloxypropylamine); (2) cationic lipids such as DLinDMA; (3) phospholipid DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) (Avanti Polar Lipids, Alabaster, Ala.); And (4) synthetic cholesterol (Sigma-Aldrich Corp., St. Louis, Mo.) (molar ratio 1.4: 57.1: 7.1: 34.3, respectively). That is, the siRNA molecule is encapsulated into the nucleic acid-lipid particle of the "1: 7" formulation as follows: 1.4% PEG2000-C-DMA; 57.1% cationic lipid; 7.1% DPPC; And 34.3% cholesterol. It is to be understood that the 1:57 formulation is a targeted formulation and that the amount of lipid present (both cationic and non-cationic lipids) and the amount of lipid conjugate present in the formulation may be different. Generally, in a 1:57 formulation, the amount of cationic lipid is 57.1 mole% ± 5 mole%, the amount of lipid conjugate is 1.4 mole% ± 0.5 mole%, the remainder of 1:57 formulation is non-cationic lipid For example, phospholipids, cholesterol, or two mixtures). The formulations are prepared using the processes described in U. S. Patent Application US 2007/0042031, all of which are incorporated herein by reference. Upon formation, the nucleic acid-lipid particles are dialyzed against PBS and filter sterilized through 0.2 mu m prior to use. The average particle size can range from 75-90 nm, where the final siRNA / lipid ratio is 0.15 (wt / wt).

Example 4 .

This example illustrates siRNA-mediated reduction of ALDH2 gene expression in human cells in vitro.

matter:

All siRNA molecules used in these studies were chemically synthesized and annealed using standard procedures.

In this example, Table A shows the aldehyde dehydrogenase 2 (ALDH2) -targeted siRNA sequences used in the experiments described in this example:

<Table A>

Way:

The nucleic acid-lipid particles consisted of the following "1:57" formulation: 1.4% PEG2000-C-DMA; 57.1% DLinDMA; 7.1% DPPC; And 34.3% cholesterol. Generally, in a 1:57 formulation, the amount of DLinDMA was 57.1 mole% ± 5 mole%, the amount of lipid conjugate was 1.4 mole% ± 0.5 mole%, and the remainder of 1:57 formulation was a non-cationic lipid , Phospholipids, cholesterol, or two mixtures) and the final siRNA / lipid ratio was about 0.11 to 0.15 (wt / wt). Upon formation of the siRNA-loaded particles, the average particle size was 85-100 nm in diameter.

Mammalian Cell Treatment and mRNA Analysis: HepG2 (human hepatocellular carcinoma) cells were maintained as adherent cultures in 96-well plates. ALDH-2-targeted or non-targeted control nucleic acid-lipid particles were added to the culture medium at siRNA concentrations of 3.91 and 15.63 [mu] g / mL; Each treatment condition was performed in duplicate wells. After incubation for about 48 h in the presence of nucleic acid-lipid particles, the cells were harvested and lysed for mRNA analysis. The human ALDH2 mRNA in the cell lysate was measured using a branched DNA assay (Penormix, Fremont, Calif., Now a subsidiary of Affymetrix, CA) and normalized to human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Respectively.

result:

In this example, Table B shows a comparison of the amount of GAPDH-normalized ALDH2 mRNA (average of two repeated test wells, ± 7.4% standard deviation) measured in the untreated culture wells set to "100.0% Gene silencing activity (mean of double wells, ± standard deviation).

<Table B>

conclusion:

All six siRNAs formulated in lipid particles silenced ALDH2 activity in human HepG2 cells in a dose-response manner. Of the six siRNAs tested, siRNA 2 showed the highest activity and siRNA 3 showed the least activity.

All US patents, US patent application publications, US patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein are incorporated herein by reference to the extent that they are inconsistent with the description of the present invention.

From the foregoing, it will be appreciated that although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention.

                         SEQUENCE LISTING <110> PROTIVA BIOTHERAPEUTICS INC.   <120> COMPOSITIONS AND METHODS FOR SILENCING ALDEHYDE        DEHYDROGENASE <130> TEKM-074 / 02WO 31118-2484 &Lt; 150 > US 61 / 599,238 <151> 2012-02-15 &Lt; 150 > US 61 / 543,700 <151> 2011-10-05 <160> 19 <170> PatentIn version 3.5 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Dicer-substracted dsRNA oligonucleotide <220> <221> modified_base <222> (1) (23) <223> n = any ribonucleic acid <220> <221> misc_feature <222> (1) <223> n is a, c, g, t or u <220> <221> modified_base <222> (24) <223> n = any deoxyribonucleic acid <400> 1 nnnnnnnnnnn nnnnnnnnnn nnnnn 25 <210> 2 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> Dicer-substracted dsRNA oligonucleotide <220> <221> modified_base <222> (1) (8) <223> n = any ribonucleic acid <220> <221> misc_feature <222> (1) (29) <223> n is a, c, g, or u <220> <221> modified_base &Lt; 222 > (9) N = any 2'OMe modified ribonucleic acid <220> <221> modified_base &Lt; 222 > (10) <223> n = any ribonucleic acid <220> <221> modified_base &Lt; 222 > (11) N = any 2'OMe modified ribonucleic acid <220> <221> modified_base (12). (12) <223> n = any ribonucleic acid <220> <221> modified_base &Lt; 222 > (13) N = any 2'OMe modified ribonucleic acid <220> <221> modified_base &Lt; 222 &gt; (14) <223> n = any ribonucleic acid <220> <221> modified_base &Lt; 222 > (15) N = any 2'OMe modified ribonucleic acid <220> <221> modified_base &Lt; 222 &gt; (16) <223> n = any ribonucleic acid <220> <221> modified_base &Lt; 222 > (17) N = any 2'OMe modified ribonucleic acid <220> <221> modified_base <222> (18). (18) <223> n = any ribonucleic acid <220> <221> modified_base <222> (19). (19) N = any 2'OMe modified ribonucleic acid <220> <221> modified_base &Lt; 222 &gt; (20) <223> n = any ribonucleic acid <220> <221> modified_base &Lt; 222 &gt; (21) N = any 2'OMe modified ribonucleic acid <220> <221> modified_base &Lt; 222 &gt; (22) <223> n = any ribonucleic acid <220> <221> modified_base &Lt; 222 &gt; (23) N = any 2'OMe modified ribonucleic acid <220> <221> modified_base &Lt; 222 &gt; (24) <223> n = any ribonucleic acid <220> <221> modified_base &Lt; 222 &gt; (25) N = any 2'OMe modified ribonucleic acid <220> <221> modified_base <222> (26) N = any ribonucleic acid, 2'OMe ribonucleic acid or absent <220> <221> modified_base <222> (29). (29) N = any ribonucleic acid or 2'OMe ribonucleic acid <400> 2 nnnnnnnnnnn nnnnnnnnnn nnnnnnnnn 29 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Aldehyde dehydrogenase 2 targeting siRNA molecule <220> <221> modified_base <222> (6) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 &gt; (13) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 > (16) <223> 2'OMe modified RNA base <400> 3 aacaagauau acugagaaat t 21 <210> 4 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Aldehyde dehydrogenase 2 targeting siRNA molecule <220> <221> modified_base &Lt; 222 &gt; (8) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 &gt; (15) <223> 2'OMe modified RNA base <220> <221> modified_base <222> (19). (19) <223> 2'OMe modified RNA base <400> 4 uuucucagua uaucuuguug g 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Aldehyde dehydrogenase 2 targeting siRNA molecule <220> <221> modified_base <222> (1) <223> 2'OMe modified RNA base <220> <221> modified_base <222> (4) (4) <223> 2'OMe modified RNA base <220> <221> modified_base (12). (12) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 &gt; (16) <223> 2'OMe modified RNA base <400> 5 gcagcaaaug agcaauaaat t 21 <210> 6 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Aldehyde dehydrogenase 2 targeting siRNA molecule <220> <221> modified_base <222> (7) (7) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 &gt; (14) <223> 2'OMe modified RNA base <220> <221> modified_base <222> (18). (18) <223> 2'OMe modified RNA base <400> 6 uuuauugcuc auuugcugcu u 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Aldehyde dehydrogenase 2 targeting siRNA molecule <220> <221> modified_base <222> (2) (2) <223> 2'OMe modified RNA base <220> <221> modified_base <222> (7) (7) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 > (13) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 &gt; (17) <223> 2'OMe modified RNA base <400> 7 ggagaaugug uaugacgaat t 21 <210> 8 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Aldehyde dehydrogenase 2 targeting siRNA molecule <220> <221> modified_base &Lt; 222 &gt; (8) &Lt; 223 &gt; 2'Ome modified RNA base <220> <221> modified_base &Lt; 222 &gt; (15) &Lt; 223 &gt; 2'Ome modified RNA base <220> <221> modified_base &Lt; 222 > (20) &Lt; 223 > 2'Ome modified RNA base <400> 8 uucgucauac acauucuccu g 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Aldehyde dehydrogenase 2 targeting siRNA molecule <220> <221> modified_base <222> (2) (2) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 &gt; (5) <223> 2'OMe modified RNA base <220> <221> modified_base (12). (12) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 &gt; (16) <223> 2'OMe modified RNA base <400> 9 cgacgccguc agcaggaaat t 21 <210> 10 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Aldehyde dehydrogenase 2 targeting siRNA molecule <220> <221> modified_base <222> (6) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 &gt; (14) <223> 2'OMe modified RNA base <220> <221> modified_base <222> (19). (19) <223> 2'OMe modified RNA base <400> 10 uuuccugcug acggcgucgu g 21 <210> 11 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Aldehyde dehydrogenase 2 targeting siRNA molecule <220> <221> modified_base <222> (2) (2) <223> 2'OMe modified RNA base <220> <221> modified_base <222> (6) <223> 2'OMe modified RNA base <220> <221> modified_base (12). (12) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 &gt; (16) <223> 2'OMe modified RNA base <400> 11 guggaugaaa cucaguuuaa g 21 <210> 12 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Aldehyde dehydrogenase 2 targeting siRNA molecule <220> <221> modified_base <222> (7) (7) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 > (9) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 > (15) <223> 2'OMe modified RNA base <400> 12 uaaacugagu uucauccacc u 21 <210> 13 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Aldehyde dehydrogenase 2 targeting siRNA molecule <220> <221> modified_base <222> (1) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 > (3) <223> 2'OMe modified RNA base <220> <221> modified_base <222> (7) (7) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 &gt; (15) <223> 2'OMe modified RNA base <400> 13 guggaugaaa cucaguuuaa g 21 <210> 14 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Aldehyde dehydrogenase 2 targeting siRNA molecule <220> <221> modified_base <222> (2) (2) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 > (5) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 > (13) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 &gt; (15) <223> 2'OMe modified RNA base <400> 14 ggaugaaacu caguuuaaga a 21 <210> 15 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Aldehyde dehydrogenase 2 targeting siRNA molecule <220> <221> modified_base &Lt; 222 > (8) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 > (13) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 > (17) <223> 2'OMe modified RNA base <400> 15 cuuaaacuga guuucaucca c 21 <210> 16 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Aldehyde dehydrogenase 2 targeting siRNA molecule <220> <221> modified_base <222> (1) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 > (5) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 > (14) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 &gt; (16) <223> 2'OMe modified RNA base <400> 16 ggaugaaacu caguuuaaga a 21 <210> 17 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Aldehyde dehydrogenase 2 targeting siRNA molecule <220> <221> modified_base <222> (2) (2) <223> 2'OMe modified RNA base <220> <221> modified_base <222> (6) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 > (14) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 &gt; (17) <223> 2'OMe modified RNA base <400> 17 cugucuucac aaaggauuug g 21 <210> 18 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Aldehyde dehydrogenase 2 targeting siRNA molecule <220> <221> modified_base &Lt; 222 > (8) <223> 2'OMe modified RNA base <220> <221> modified_base (12). (12) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 > (15) <223> 2'OMe modified RNA base <220> <221> modified_base <222> (19). (19) <223> 2'OMe modified RNA base <400> 18 aaauccuuug ugaagacagc u 21 <210> 19 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Aldehyde dehydrogenase 2 targeting siRNA molecule <220> <221> modified_base <222> (2) (2) <223> 2'OMe modified RNA base <220> <221> modified_base <222> (4) (4) <223> 2'OMe modified RNA base <220> <221> modified_base <222> (7) (7) <223> 2'OMe modified RNA base <220> <221> modified_base &Lt; 222 &gt; (15) <223> 2'OMe modified RNA base <400> 19 cugucuucac aaaggauuug g 21

Claims (66)

Wherein the interfering RNA comprises an interfering RNA that silences the expression of an aldehyde dehydrogenase (ALDH) gene, wherein the interfering RNA comprises a sense strand and a complementary antisense strand and a double stranded region of about 15 to about 60 nucleotides in length. . The composition of claim 1, wherein the interfering RNA is a double stranded RNA (dsRNA) selected from the group consisting of siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, pre-miRNA, and combinations thereof. 3. The composition of claim 1, wherein the interfering RNA comprises a double-stranded region of about 19 to about 25 nucleotides in length. 2. The composition of claim 1 wherein the interfering RNA is chemically synthesized. 2. The composition of claim 1, wherein the interfering RNA comprises a 3 ' overhang at one or both strands of the interfering RNA. 4. The composition of claim 1, wherein the interfering RNA comprises at least one modified nucleotide in the double-stranded region. 7. The composition of claim 6, wherein less than about 30% of the nucleotides in the double-stranded region comprise modified nucleotides. 7. The composition of claim 6, wherein the modified nucleotide is a 2'-O-methyl (2'OMe) nucleotide. 9. The composition of claim 8, wherein the 2'OMe nucleotide comprises a 2'OMe-guanosine nucleotide, a 2'OMe-uridine nucleotide, or a mixture thereof. The composition of claim 1, further comprising a pharmaceutically acceptable carrier. 11. The composition according to any one of claims 1 to 10, wherein the interfering RNA silences aldehyde dehydrogenase (ALDH2) gene expression. (a) the interfering RNA of claim 1;
(b) cationic lipids; And
(c) non-cationic lipid
&Lt; / RTI &gt; 12. The composition of claim 11, wherein the cationic lipid is selected from the group consisting of 1,2-dilinoleoyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy- 1,2-di-gamma -linolenyloxy-N, N-dimethylaminopropane (γ-DLenDMA), salts thereof, or mixtures thereof. 13. The composition of claim 12 wherein the cationic lipid is selected from the group consisting of 2,2-dirinoleyl-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (DLin- Dirinoleyl-4-dimethylaminomethyl- [1,3] -dioxolane (DLin-K-DMA), salts thereof, or mixtures thereof. 13. The method of claim 12, wherein the cationic lipid is selected from the group consisting of (6Z, 9Z, 28Z, 31Z) -heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butanoate -M-C3-DMA), dirinoleylmethyl-3-dimethylaminopropionate (DLin-M-C2-DMA), salts thereof, or mixtures thereof. 13. The nucleic acid-lipid particle of claim 12, wherein the non-cationic lipid is a phospholipid. 13. The nucleic acid-lipid particle of claim 12, wherein the non-cationic lipid is cholesterol or a derivative thereof. 13. The nucleic acid-lipid particle of claim 12, wherein the non-cationic lipid is a mixture of a phospholipid and cholesterol or a derivative thereof. 19. The nucleic acid-lipid particle according to claim 16 or 18, wherein the phospholipid comprises dipalmitoyl phosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), or a mixture thereof. 13. The nucleic acid-lipid particle of claim 12, wherein the non-cationic lipid is a mixture of DPPC and cholesterol. 13. The nucleic acid-lipid particle of claim 12, further comprising a conjugated lipid that inhibits aggregation of particles. 22. The nucleic acid-lipid particle according to claim 21, wherein the binding lipid for inhibiting aggregation of particles is a polyethylene glycol (PEG) -lipid conjugate. 23. The method of claim 22 wherein the PEG-lipid conjugate is selected from the group consisting of PEG-diacylglycerol (PEG-DAG) conjugate, PEG-dialkyloxypropyl (PEG-DAA) conjugate, PEG-phospholipid conjugate, PEG- , &Lt; / RTI &gt; and mixtures thereof. 23. The nucleic acid-lipid particle of claim 22, wherein the PEG-lipid conjugate is a PEG-DAA conjugate. 25. The method of claim 24, PEG-DAA conjugate is PEG- didecyl oxy profile (C ₁₀₎ conjugate, PEG- dilauryl oxy profile (C ₁₂₎ conjugate, PEG- dimyristyl oxy profile (C ₁₄₎ conjugate, PEG- palmityl di-oxy-propyl (C ₁₆₎ conjugate, PEG- distearyl oxy profile (C ₁₈₎ conjugate, and a is selected from the group consisting of mixtures of these nucleic acid-lipid particle. 22. The nucleic acid-lipid particle according to claim 21, wherein the bonding lipid that inhibits aggregation of particles is a polyoxazoline (POZ) -lipid conjugate. 27. The nucleic acid-lipid particle of claim 26, wherein the POZ-lipid conjugate is a POZ-DAA conjugate. 13. The nucleic acid-lipid particle of claim 12, wherein the interfering RNA is fully encapsulated within the particle. 13. The nucleic acid-lipid particle of claim 12, wherein the lipid: interfering RNA mass ratio of the particles is from about 5: 1 to about 15: 1. 13. The nucleic acid-lipid particle of claim 12, wherein the median diameter of the particles is from about 30 nm to about 150 nm. 13. The nucleic acid-lipid particle of claim 12, wherein the cationic lipid comprises from about 50 mole% to about 65 mole% of the total lipid present in the particle. 13. The composition of claim 12, wherein the non-cationic lipid comprises a phospholipid and a mixture of cholesterol or derivatives thereof wherein the phospholipid comprises from about 4 mole percent to about 10 mole percent of the total lipid present in the particle, Of the total lipid present in the particle occupies about 30 mole% to about 40 mole% of the total lipid present in the particle. 33. The method of claim 32, wherein the phospholipid comprises from about 5 mole percent to about 9 mole percent of the total lipid present in the particle and wherein the cholesterol or derivative thereof comprises from about 32 mole percent to about 37 mole percent of the total lipid present in the particle Nucleic acid-lipid particles. 22. The nucleic acid-lipid particle of claim 21, wherein the cohesive lipid that inhibits aggregation of the particles comprises from about 0.5 mole percent to about 2 mole percent of the total lipid present in the particles. 14. A pharmaceutical composition comprising the nucleic acid-lipid particle of claim 12 and a pharmaceutically acceptable carrier. 12. A method of introducing into a cell an interfering RNA that silences ALDH gene expression, comprising contacting the cell with a nucleic acid-lipid particle of claim 12. 37. The method of claim 36, wherein the cell is in a mammal. 37. The method of claim 36, wherein the cells are contacted by administering the particles via a systemic route to the mammal. 37. The method of claim 36, wherein the mammal is a human. 37. The method of claim 36, wherein the mammal is diagnosed with alcoholism. 37. The method of claim 36, wherein the interfering RNA silences ALDH2 gene expression in the cell. 19. A method of silencing ALDH gene expression in a mammal comprising administering to said mammal a nucleic acid-lipid particle of claim 12 in need of silencing of ALDH gene expression. 43. The method of claim 42, wherein the particles are administered via a systemic route. 43. The method of claim 42, wherein the mammal is a human. 45. The method of claim 44, wherein the human is afflicted with alcohol. 43. The method of claim 42, wherein administration of the particle reduces the level of ALDH RNA in the mammal by at least about 50% relative to the level of ALDH RNA in the absence of the particle. 43. The method of claim 42, wherein the nucleic acid-lipid particle silences ALDH2 gene expression. A method of treating and / or ameliorating one or more symptoms associated with alcoholism in a human, comprising administering to a human a therapeutically effective amount of the nucleic acid-lipid particle of claim 12. 49. The method of claim 48, wherein the particles are administered via a systemic route. 49. The method of claim 48, wherein the human is in an alcoholic state. 49. The method of claim 48, wherein the interfering RNA of the nucleic acid-lipid particle inhibits the expression of the ALDH2 gene in a human. Comprising administering to a mammal in need of inhibition of ALDH expression a therapeutically effective amount of the nucleic acid-lipid particle of claim 12. 20. A method of inhibiting ALDH expression in a mammal, 53. The method of claim 52, wherein the particles are administered via a systemic route. 53. The method of claim 52, wherein the mammal is a human. 55. The method of claim 54, wherein the human is in an alcoholic state. 53. The method of claim 52, wherein administration of the particle reduces ALDH gene expression in the mammal by at least about 50% relative to ALDH gene expression in the absence of the particle. 53. The method of claim 52, wherein the expression of ALDH2 is suppressed in the mammal. A method of preventing and / or treating alcohol intoxication in a human, comprising administering to a human a therapeutically effective amount of the nucleic acid-lipid particle of claim 12. 59. The method of claim 58, wherein the particles are administered via a systemic route. 59. The method of claim 58, wherein the human is afflicted with alcohol. 59. The method of claim 58, wherein the interfering RNA of the nucleic acid-lipid particle inhibits the expression of the ALDH2 gene in a human. A double-stranded siRNA molecule selected from the group consisting of an identifier 1, an identifier 2, an identifier 3, an identifier 4, an identifier 5, and an identifier 6 shown in Table A of Example 4 of the present application. Single-stranded RNA molecule selected from the single-stranded sense-strand molecule set forth in Table A of Example 4 herein. Single stranded RNA molecule selected from the single-stranded antisense strand molecules set forth in Table A of Example 4 herein. The use of siRNA molecules of the invention for inhibiting ALDH gene expression in living cells. Use of a pharmaceutical composition of the invention for inhibiting ALDH gene expression in living cells.